CN105814201A - Protein comprised by linking by linker multiple domains having affinity for proteins having fc part of immunoglobulin G (IgG) - Google Patents

Abstract

The purpose of the present invention is to provide a novel protein, which have such excellent pH responsiveness that the antibody binding property to the Fc region of the immunoglobulin in the neutral region is improved and the binding property to the Fc region of the immunoglobulin in the weakly acidic region is much decreased when compared to the wild-type proteins such as protein G and protein A that have affinity for the protein comprising the Fc region of the immunoglobulin G (IgG), and a mutant-type protein (an improved protein) comprising their domain mutant. The present invention relates to a protein constituted by linking plural domains having affinity for a protein comprising the Fc region of the immunoglobulin G (IgG) with linkers, wherein an extended length to the maximum of the linker is 80 -240 angstrom.

Description

Bonding with a linker is useful for proteins with the Fc portion of immunoglobulin G (IgG) affinity between multiple domains of proteins

technical field

The present invention relates to a protein formed by bonding a plurality of domains having an affinity for a protein having an Fc portion of immunoglobulin G (IgG) with a linker, in particular characterized in that the linker has a maximum elongation length of about 80Å ~ 240Å of the aforementioned proteins, etc.

Background technique

Conventionally, the purification of proteins including antibodies has been an important subject in biochemical research, and various techniques such as affinity chromatography, gel filtration chromatography, and ion exchange chromatography are known. Affinity chromatography is a method of purifying a target protein by utilizing its specific affinity with the target protein. This method can easily and selectively recover proteins, and its affinity is very strong. Therefore, in order to dissociate the proteins adsorbed on the chromatography filler, generally speaking, it is necessary to use an acidic buffer solution with a pH of about 2.5 for elution. Under such strongly acidic conditions, activity reduction such as protein denaturation is likely to occur, and purification under milder conditions is required.

Protein G, which is a protein derived from Streptococcus, is a membrane protein present in the cell membrane of Streptococcus genus Streptococcus, and is known to have specificity for the Fc region of immunoglobulin G (one of antibodies) Binding activity (Non-Patent Document 1, Patent Document 1). Protein G is a multi-domain membrane protein composed of multiple domains, and it is a part of the cell membrane that exhibits binding activity to proteins having the Fc region of immunoglobulin G (hereinafter referred to as "antibody binding activity") ectodomain (Non-Patent Document 2). For example, in the case of protein G derived from the G148 strain shown in FIG. 1 , three domains, B1, B2, and B3 (also referred to as C1, C2, and C3 domains in literature), exhibit antibody-binding activity. In addition, there are three antibody-binding domains in protein G of the GX7805 strain, and two antibody-binding domains in protein G of GX7809. These are known to be small proteins of approximately 60 amino acids, showing high identity between their amino acid sequences). It is also known that antibody-binding activity is maintained even when protein G is cleaved and each domain is individually isolated (Non-Patent Document 3).

With regard to the cell extramembrane domain of protein G, at present, various protein G cell extramembrane domain-containing products utilizing its selective antibody binding activity (for example, carriers for affinity chromatography for antibody purification (Patent Document 3, 4), inspection reagents for detecting antibodies, research reagents, etc.) have been listed. It is known that the binding force between the extracellular domain of protein G and antibodies is high in the neutral to weakly acidic region and low in the strongly acidic region (Non-Patent Document 4). Therefore, for the purpose of isolating, recovering, and purifying antibodies, first, neutralize a sample solution containing antibodies such as serum, and bring them into contact with water-insoluble beads such as beads immobilized with the extracellular domain of protein G. A solid support that selectively adsorbs antibodies. Thereafter, components other than antibodies were removed by washing with a neutral to weak acid solution (pH 5 to 8). Finally, usually, a strongly acidic solution of pH 2.4 to 3.5 is added to detach the antibody from the immobilized protein G, and the antibody is eluted together with the strongly acidic solution (Patent Document 3). Thus, antibodies can be separated, recovered, and purified with high purity.

However, when an antibody is placed in a strongly acidic solution of pH 2.4 to 3.5, it deteriorates due to denaturation and aggregation, etc., and may lose its original function depending on the type of antibody (Non-Patent Document 4). In order to prevent these situations, it is attempted to treat in a weakly acidic region with a pH higher than pH 2.4 to 3.5. However, due to the strong binding force between the extracellular domain of protein G and the antibody, the antibody is not washed from protein G in the weakly acidic region. Take off, not enough recovery amount can be obtained. On the other hand, it is known that the protein G cell extramembrane domain also binds to Fab (Non-Patent Document 2), and that one antibody molecule can bind to the protein G cell extramembrane domain in two regions, the Fc region and the Fab region. In such a bonded state, the antibody and the extracellular domain of protein G cannot be easily dissociated, making it difficult to recover the antibody.

So far, the inventors of the present invention have developed cell membranes made of protein G that have thermal stability, chemical stability to denaturants, and resistance to proteolytic enzymes (these properties are collectively referred to as "protein stability") Modified proteins composed of ectodomain mutants (Patent Document 5 and Patent Document 6), and those with reduced binding to the Fc region of immunoglobulin and/or to the Fab region in the weakly acidic region have also been developed Modified protein (Patent Document 7). However, each of these modified proteins contained only one domain showing antibody-binding activity.

Furthermore, the inventors of the present invention developed a protein composed of a tandem polymer of the above-mentioned modified protein (Patent Document 8). For such a protein, the binding property to the weakly acidic region of the Fc region of human immunoglobulin G of different subclasses such as IgG1 and IgG3 compared with the tandem multimer of the wild-type protein G・B1 domain Even lower, in a chromatography column for protein separation and purification filled with the capture agent of the present invention containing the protein, the captured protein can be more easily eluted in a weakly acidic region (about pH 4 to 5) without denaturation. Human immunoglobulin G.

In addition, protein A, which is a protein derived from Staphylococcus aureus, is known to have specific binding activity to the Fc region of immunoglobulin G (a type of antibody) (Non-Patent Document 5, Non-Patent Document 6). Protein A is a multi-domain membrane protein composed of multiple domains, and it is a part of the cell extramembrane domain that exhibits binding activity to proteins having an immunoglobulin-Fc region (hereinafter referred to as antibody binding activity) (Non-Patent Document 6). For example, in the case of protein A derived from the NCTC8325 strain, five domains of E, D, A, B, and C exhibit antibody-binding activity. These are small proteins of almost 60 amino acids showing high identity between their amino acid sequences. In addition, it is known that antibody-binding activity is maintained even when protein A is cleaved and each domain is individually isolated (Non-Patent Document 7). On the other hand, the Z domain is an artificial protein synthesized based on the sequence of the B domain (Non-Patent Document 7), and differs from the B domain by 2 amino acid residues. It is known that the two substitutions, Ala1Val and Gly29Ala, stabilize the structure without losing antibody-binding property, and the thermal denaturation temperature becomes 90° C. or higher (Non-Patent Document 7).

For the extracellular domains (E, D, A, B, C) and the Z domain of protein A, a variety of preparations utilizing their selective antibody binding activity are now on the market (e.g. and chromatography carriers, inspection reagents for detecting antibodies, research reagents, etc.). It is known that the binding force between the extracellular domain of protein A and the antibody is high in the neutral region and low in the strongly acidic region (Non-Patent Document 8). Therefore, for the purpose of isolating, recovering, and purifying antibodies, first, a sample solution containing antibodies such as serum is brought into a neutral state, and brought into contact with a water-insoluble solid such as beads immobilizing the extracellular domain of protein A. phase support, which selectively adsorbs antibodies. Thereafter, components other than antibodies were removed by washing with a neutral solution of pH 7. Finally, a strongly acidic solution at pH 3.0 is usually added to desorb the antibody from the immobilized protein A and elute together with the strongly acidic solution. Thus, antibodies can be separated, recovered, and purified with high purity.

Regarding protein A, the present inventors have so far developed a mutant protein containing a mutant of the extracellular domain of protein A and an antibody complement (Patent Document 9) that has improved ease of dissociation in the weakly acidic region, and a protein in the acidic region. A mutant protein containing a mutant protein A extramembrane domain with reduced affinity and an antibody complement (Patent Document 10).

prior art literature

patent documents

Patent Document 1: JP-03-501801 Publication

Patent Document 2: Japanese Patent No. 2764021

Patent Document 3: Japanese Patent Application Laid-Open No. 03-128400

Patent Document 4: Japanese Patent Laid-Open No. 2003-088381

Patent Document 5: Japanese Patent Laid-Open No. 2009-95322

Patent Document 6: Japanese Patent Laid-Open No. 2009-118749

Patent Document 7: Japanese Patent Laid-Open No. 2009-297018

Patent Document 8: International Publication No. 2013/018880

Patent Document 9: Japanese Patent Laid-Open No. 2010-81866

Patent Document 10: International Publication No. 2012/165544

non-patent literature

Non-Patent Document 1: Bjorck L, Kronvall G. (1984) Purification and some properties of streptococcal protein G, a novel IgG-binding reagent. JImmunol. 133, 969-974.

Non-Patent Document 2: Boyle M. D.P., Ed. (1990) Bacterial Immunoglobulin Binding Proteins. Academic Press, Inc., San Diego, CA, USA.

Non-Patent Document 3: Gallagher T, Alexander P, Bryan P, Gilliland GL. (1994) Twocrystal structures of the B1 immunoglobulin-binding domain of streptococcalprotein G and comparison with NMR. Biochemistry 19, 4721-4729.

Non-Patent Document 4: Gagnon P. (1996) Purification Tools for Monoclonal Antibodies, Validated Biosystems Inc., Tucson, AZ, USA.

Non-Patent Document 5 : Forsgren A and Sjoquist J (1966) "Protein A" from S.Aureus. J Immunol. 97, 822-827.

Non-Patent Document 6: Boyle M. D.P., Ed. (1990) Bacterial Immunoglobulin Binding Proteins. Academic Press, Inc., San Diego, CA, USA.

Non-Patent Document 7: Tashiro M, Montelione GT. (1995) Structures of bacterial immunoglobulin-bindingdomains and their complexes with immunoglobulins. CurrOpin Struct Biol. 5, 471-481.

Non-Patent Document 8: Gagnon P. (1996) Purification Tools for Monoclonal Antibodies, Validated Biosystems Inc., Tucson, AZ, USA.

Non-Patent Document 9: Evers TH, van Dongen EM, Faesen AC, Meijer EW, Merkx M., (2006) Quantitative understanding of the energy transfer between fluorescent proteins connected via flexible peptide linkers, Biochemistry. Nov 7;45(44):13183 -92.

Contents of the invention

The problem to be solved by the invention

The problem to be solved by the present invention is to provide wild-type proteins such as protein G and protein A containing domains having an affinity for proteins having the Fc portion of immunoglobulin G (IgG), and mutations containing mutants thereof. The antibody-binding activity of the Fc region of the immunoglobulin possessed by the type protein (modified protein) is improved in the neutral region, and further, it has the binding property to the Fc region of the immunoglobulin in the weakly acidic region compared with these proteins Novel proteins with more reduced excellent pH responsiveness, etc.

Furthermore, a capture agent for an antibody, immunoglobulin G, or a protein (antibody, etc.) having a Fab region of immunoglobulin G, which is characterized in that the protein is immobilized and useful as an affinity chromatography filler for antibody purification, A chromatography column for protein separation and purification filled with the capturing agent, particularly an affinity chromatography column for antibody purification.

The inventors of the present invention found that the above-mentioned problems can be solved by adjusting the length of the linker linking each domain in the wild-type protein and its mutant protein to an appropriate range, and completed the present invention.

That is, each embodiment of the present invention is as follows.

[Embodiment 1]

A protein in which a plurality of domains having an affinity for a protein having an Fc portion of immunoglobulin G (IgG) are bonded by a linker, wherein the linker has a maximum elongation length of 80 Å to 240 Å.

[Embodiment 2]

The protein according to embodiment 1, wherein the linker is a polypeptide linker.

[Embodiment 3]

The protein according to Embodiment 2, wherein the linker consists of a polypeptide of 22 to 66 amino acid residues.

[Embodiment 4]

The protein according to Embodiment 3, wherein the amino acid sequence of the linker consists of 4-10 repeating units of GlyGlySerGlyGlySer.

[Embodiment 5]

The protein according to Embodiment 4, wherein the amino acid sequence of the linker consists of 6-10 repeating units of GlyGlySerGlyGlySer.

[Embodiment 6]

The protein according to any one of embodiments 1-5, wherein the domain is derived from protein G or protein A.

[Embodiment 7]

The protein according to Embodiment 6, wherein the domain is a mutant of any one of the domains of wild-type protein G·B1, B2, or B3.

[Embodiment 8]

The protein according to any one of embodiments 1 to 7, wherein the domains are identical to each other.

[Embodiment 9]

The protein according to any one of embodiments 1 to 8, wherein the two domains are a linked dimer.

[Embodiment 10]

The protein according to any one of embodiments 7-9, wherein the domain mutant is the following mutant protein:

A mutant protein of the wild-type protein G·B1 domain protein, which consists of the amino acid sequence shown in (a) below, or one or more amino acid residues in the amino acid sequence shown in (a) are deleted, Substituted, inserted or added amino acid sequence composition, has binding activity to the Fc region of immunoglobulin G, and at least has binding activity to the Fab region of immunoglobulin G and / or reduced binding activity to the Fc region in the weakly acidic region:

(In the above amino acid sequences, X35 represents Asn or Lys, X36 represents Asp or Glu, X37 represents Asn, His, or Leu, X47 represents Asp or Pro, X48 represents Ala, Lys or Glu, X22 represents Asp or His, respectively, in the above amino acid sequences, X25 represents Thr or His, X32 represents Gln or His, X40 represents Asp or His, X42 represents Glu or His, X11 represents Thr or Arg, and X17 represents Thr or Ile. However, X35 is Asn or Lys and X36 is Asp simultaneously. or Glu, X37 is Asn or Leu, X47 is Asp or Pro, X48 is Ala, Lys or Glu, X22 is Asp, X25 is Thr, X32 is Gln, X40 is Asp, X42 is Glu, X11 is Thr, and X17 is In the case of Thr.).

[Embodiment 11]

The protein according to any one of embodiments 7-9, wherein the domain mutant is the following mutant protein:

A mutant protein of the wild-type protein G.B2 domain protein, which consists of the amino acid sequence shown in (b) below, or one or more amino acid residues in the amino acid sequence shown in (b) are deleted, Substituted, inserted or added amino acid sequence composition, has binding activity to the Fc region of immunoglobulin G, and at least has binding activity to the Fab region of immunoglobulin G and / or reduced binding activity to the Fc region in the weakly acidic region:

(In the above amino acid sequences, X35 represents Asn or Lys, X36 represents Asp or Glu, X37 represents Asn, His, or Leu, X47 represents Asp or Pro, X48 represents Ala, Lys or Glu, X22 represents Asp or His, respectively, in the above amino acid sequences, X25 represents Thr or His, X32 represents Gln or His, X40 represents Asp or His, X42 represents Glu or His, X11 represents Thr or Arg, and X17 represents Thr or Ile. However, X35 is Asn or Lys and X36 is Asp simultaneously. or Glu, X37 is Asn or His, X47 is Asp or Pro, X48 is Ala, Lys or Glu, X22 is Asp, X25 is Thr, X32 is Gln, X40 is Asp, X42 is Glu, X11 is Thr, and X17 is In the case of Thr.).

[Embodiment 12]

The protein according to any one of embodiments 7-9, wherein the domain mutant is the following mutant protein:

A mutant protein of the wild-type protein G.B3 domain protein, which consists of the amino acid sequence shown in (c) below, or one or more amino acid residues in the amino acid sequence shown in (c) are deleted, Substituted, inserted or added amino acid sequence composition, has binding activity to the Fc region of immunoglobulin G, and at least has binding activity to the Fab region of immunoglobulin G and / or reduced binding activity to the Fc region in the weakly acidic region:

(In the above amino acid sequences, X35 represents Asn or Lys, X36 represents Asp or Glu, X37 represents Asn, His, or Leu, X47 represents Asp or Pro, X48 represents Ala, Lys or Glu, X22 represents Asp or His, respectively, in the above amino acid sequences, X25 represents Thr or His, X32 represents Gln or His, X40 represents Asp or His, X11 represents Thr or Arg, and X17 represents Thr or Ile. However, X35 is Asn or Lys, X36 is Asp or Glu, and X37 is Asn simultaneously. or His, X47 is Asp or Pro, X48 is Ala, Lys or Glu, X22 is Asp, X25 is Thr, X32 is Gln, X40 is Asp, X11 is Thr, and X17 is Thr.).

[Embodiment 13]

The protein according to any one of embodiments 7-9, wherein the domain mutant is the following mutant protein:

A mutant protein of the wild-type protein G·B1 domain protein, which consists of the amino acid sequence shown in (d) below, or one or more amino acid residues in the amino acid sequence shown in (d) are deleted, Substituted, inserted, or added amino acid sequences have binding activity to the Fc region of immunoglobulin G, and, compared with wild-type protein G·B1 domain protein, binding activity to the Fab region of immunoglobulin G and / or reduced binding activity to the Fc region in the weakly acidic region:

(in the above amino acid sequences, respectively, X22 represents Asp or His, X25 represents Thr or His, X32 represents Gln or His, X40 represents Asp or His, X42 represents Glu or His, X11 represents Thr or Arg, X17 represents Thr or Ile However, the case where X22 is Asp, X25 is Thr, X32 is Gln, X40 is Asp, X42 is Glu, X11 is Thr, and X17 is Thr simultaneously is excluded.).

[Embodiment 14]

The protein according to any one of embodiments 7-9, wherein the domain mutant is the following mutant protein:

A mutant protein of the wild-type protein G·B2 domain protein, which consists of the amino acid sequence shown in (e) below, or one or more amino acid residues in the amino acid sequence shown in (e) are deleted, Substituted, inserted or added amino acid sequence composition, has binding activity to the Fc region of immunoglobulin G, and has binding activity to the Fab region of immunoglobulin G and/or compared with wild-type protein G·B2 domain protein Or the binding activity of the Fc region in the weakly acidic region is reduced:

(in the above amino acid sequences, respectively, X22 represents Asp or His, X25 represents Thr or His, X32 represents Gln or His, X40 represents Asp or His, X42 represents Glu or His, X11 represents Thr or Arg, X17 represents Thr or Ile However, the case where X22 is Asp, X25 is Thr, X32 is Gln, X40 is Asp, X42 is Glu, X11 is Thr, and X17 is Thr simultaneously is excluded.).

[Embodiment 15]

The protein according to any one of embodiments 7-9, wherein the domain mutant is the following mutant protein:

A mutant protein of the wild-type protein G.B3 domain protein, which consists of the amino acid sequence shown in (f) below, or one or more amino acid residues in the amino acid sequence shown in (f) are deleted, Substituted, inserted or added amino acid sequence composition, has binding activity to the Fc region of immunoglobulin G, and has binding activity to the Fab region of immunoglobulin G and/or compared with the wild-type protein G·B3 domain protein Or the binding activity of the Fc region in the weakly acidic region is reduced:

(In the above amino acid sequences, X22 represents Asp or His, X25 represents Thr or His, X32 represents Gln or His, X40 represents Asp or His, X11 represents Thr or Arg, X17 represents Thr or Ile, respectively. However, simultaneously X22 is Asp, X25 is Thr, X32 is Gln, X40 is Asp, X11 is Thr, and X17 is Thr.).

[Embodiment 16]

The protein according to any one of embodiments 7-9, wherein the domain mutant is the following mutant protein:

A mutant protein of the wild-type protein G·B1 domain protein, which consists of the amino acid sequence shown in (g) below, or one or more amino acid residues in the amino acid sequence shown in (g) are deleted, Substituted, inserted or added amino acid sequences have binding activity to the Fc region of immunoglobulin G, and compared with the wild-type protein G B1 domain protein, the binding activity to the Fc region in the weakly acidic region is reduced:

(In the above amino acid sequences, X22 represents Asp or His, X25 represents Thr or His, X32 represents Gln or His, X40 represents Asp or His, X42 represents Glu or His, respectively. However, X22 is Asp and X25 is The case where Thr, X32 is Gln, X40 is Asp, and X42 is Glu.).

[Embodiment 17]

The protein according to any one of embodiments 7-9, wherein the domain mutant is the following mutant protein:

A mutant protein of the wild-type protein G·B2 domain protein, which consists of the amino acid sequence shown in (h) below, or one or more amino acid residues in the amino acid sequence shown in (h) are deleted, Substituted, inserted or added amino acid sequences have binding activity to the Fc region of immunoglobulin G, and compared with the wild-type protein G B2 domain protein, the binding activity to the Fc region in the weakly acidic region is reduced:

(In the above amino acid sequences, X22 represents Asp or His, X25 represents Thr or His, X32 represents Gln or His, X40 represents Asp or His, X42 represents Glu or His, respectively. However, X22 is Asp and X25 is The case where Thr, X32 is Gln, X40 is Asp, and X42 is Glu.).

[Embodiment 18]

The protein according to any one of embodiments 7-9, wherein the domain mutant is the following mutant protein:

A mutant protein of the wild-type protein G.B3 domain protein, which consists of the amino acid sequence shown in (i) below, or one or more amino acid residues in the amino acid sequence shown in (i) are deleted, Substituted, inserted or added amino acid sequences have binding activity to the Fc region of immunoglobulin G, and compared with the wild-type protein G B3 domain protein, the binding activity to the Fc region in the weakly acidic region is reduced:

(In the above amino acid sequences, X22 represents Asp or His, X25 represents Thr or His, X32 represents Gln or His, and X40 represents Asp or His, respectively. However, simultaneously X22 is Asp, X25 is Thr, X32 is Gln, and X40 is the case for Asp.).

[Embodiment 19]

The protein according to any one of embodiments 7-9, wherein the domain mutant is a mutant protein having the following amino acid sequence (SEQ ID NO: 19):

[Embodiment 20]

A protein which is a fusion protein consisting of an amino acid sequence in which the amino acid sequence of the protein according to any one of Embodiments 1 to 19 is linked with the amino acid sequence of another protein.

[Embodiment 21]

The protein according to embodiment 20, which has the amino acid sequence shown in any one of SEQ ID NO:41-45.

[Embodiment 22]

A nucleic acid encoding the protein of any one of embodiments 1-21.

[Embodiment 23]

A recombinant vector containing the nucleic acid according to embodiment 22.

[Embodiment 24]

A transformant into which the recombinant vector according to Embodiment 23 has been introduced.

[Embodiment 25]

A capture agent for immunoglobulin G or a protein having an Fc region or Fab region of immunoglobulin G, characterized in that the protein according to any one of embodiments 1 to 21 is immobilized on a water-insoluble solid support.

[Embodiment 26]

Affinity chromatography for purification of immunoglobulin G or a protein having an Fc region or Fab region of immunoglobulin G, comprising the capture agent according to Embodiment 25.

[Embodiment 27]

A method of purifying immunoglobulin G or a protein having an Fc region or Fab region of immunoglobulin G, using the affinity chromatography for purification described in Embodiment 26.

The effect of the invention

It has been found by the present invention that the length of maximal elongation of linkers between domains that bond to proteins having an affinity (specific binding activity) for the Fc portion of immunoglobulin G (IgG) is at about 80 Å Range of ~240Å, wild-type proteins such as protein G and protein A containing peptides consisting of about 22 to 66 amino acids between the domain and existing mutant proteins containing mutants of their domains (modified protein), the antibody-binding activity to the Fc region of immunoglobulin in the neutral region is significantly improved, and further, compared with these proteins, the binding property to the Fc region of immunoglobulin in the weakly acidic region is stronger reduced, and successfully achieved excellent pH responsiveness.

As a result, by using the protein of the present invention as a capture agent, it is surprisingly possible to dissociate and wash antibodies under milder conditions in a weakly acidic region while maintaining neutral antibody binding to immunoglobulins such as human IgG. Deimmunoglobulin, even for immunoglobulins that are easily denatured in acid, can be recovered by affinity purification in a pH region around neutral without the risk of denaturation by acid.

Brief description of the drawings

[ Fig. 1 ] shows the structure of protein G extracellular domain mutant tandem dimers (PG2LL10, PG2LL7, PG2LL6, PG2LL5, PG2LL4, PG2LL1).

[ Fig. 2-1 ] shows the results of SPR measurement under the same mass protein immobilization conditions showing the comparison of protein G extracellular domain mutant tandem dimers (PG2LL5, PG2LL4, PG2LL1).

[ Fig. 2-2 ] shows the results of SPR measurement under the same mass protein immobilization conditions showing the comparison of protein G extracellular domain mutant tandem dimers (PG2LL10, PG2LL7, PG2LL6).

[ Fig. 3] Fig. 3 is a graph showing comparison of antibody binding rates of protein G extracellular domain mutant tandem dimers (PG2LL10, PG2LL7, PG2LL6, PG2LL5, PG2LL4, PG2LL1). The antibody binding rate was calculated from the SPR of PG2LL4-6 when the SPR reaction of PGLL1 was 0% and the SPR reaction of PG2LL10 was 100% 500 seconds after the end of antibody addition (Relative binding stability (%)) ).

[ Fig. 4 ] shows the results of pH gradient affinity chromatography using the recombinant PG-immobilized column of the present invention.

detailed description

The protein of the present invention is a protein in which a plurality of structural domains having an affinity for a protein having an Fc portion of immunoglobulin G (IgG) are bonded by a linker, and is characterized in that the maximum elongation length of the linker is controlled by Adjust to be in the range of about 80Å to 240Å.

The chemical structure of the linker contained in the protein of the present invention is not particularly limited, for example, a polypeptide linker composed of amino acids is preferable. The type and sequence of amino acids constituting the peptide linker are not particularly limited. For example, it may be composed of repeating units of peptides composed of a plurality of amino acids. A suitable example of such a repeating unit (unit) includes "GlyGlySerGlyGlySer". This sequence is an amino acid sequence commonly used in the design process of proteins such as single-chain antibodies in this technical field, and has favorable properties such as low immunogenicity and little influence on living bodies.

The length of the maximum stretchable structure of each unit "GlyGlySerGlyGlySer" is about 21.6 Å (the "maximum stretchable length" is calculated based on the records of Maruzen Co., Ltd. on page 34 of the 19th edition of Harper Biochemistry). Therefore, When it is used as a repeating unit, the maximum elongation length of the peptide linker consisting of 4 to 11 units (the total number of amino acids is 24 to 66) becomes about 86 Å to 240 Å, and the number of this unit is 4 to 10 The length of the maximum elongation of the peptide linker (the total number of amino acids is 24 to 60) becomes about 86 Å to 216 Å.

Therefore, the number of amino acids constituting the peptide linker contained in the protein of the present invention is usually about 22 to 66, preferably about 24 to 60, depending on the type of amino acid (chemical structure). A preferred example thereof includes a peptide linker composed of 4 to 10, more preferably 6 to 10, repeating units of the unit "GlyGlySerGlyGlySer".

The domain contained in the protein of the present invention is not particularly limited in terms of its type, structure, source, etc. as long as it has an affinity for a protein having an Fc portion of immunoglobulin G (IgG), and any domain known to those skilled in the art can be used. . Preferable examples thereof include domains derived from protein G or protein A, for example, their wild-type domains, and mutants that alter any of the domains of wild-type protein G, B1, B2, or B3. A domain mutant derived from a portion of the amino acid sequence of the wild-type domain. Preferred examples of such domain mutants are more specifically disclosed in the present specification. Mutations in the amino acid sequence of such a modified protein can be carried out by any method known to those skilled in the art, as described in Examples of Patent Document 8, for example.

Furthermore, the number of domains contained in the protein of the present invention can be appropriately selected according to the use of the protein, the type of domain, and the like. For example, dimers, trimers, tetramers, or pentamers may be used. Furthermore, the domains contained in the protein of the present invention may be different from or identical to each other.

It should be noted that the distance between the two domains of protein G in the complex in which the Fc region of the antibody interacts with protein G at a bivalency can be obtained from a known structure drawing software (for example, ViewerLite 5.0 (Accelrys Inc)). (References: Sauer-Eriksson AE, Kleywegt GJ, Uhlen M, Jones TA. Crystal structure of the C2 fragment of streptococcal protein G in complex with the Fc domain of human IgG. Structure. 1995 Mar 15;3(3): 265-78.), the distance between the domains of protein G is estimated to be about 66 Å. Therefore, when two domains interact bivalently, a linker having a length greater than this distance is necessary. In comparison, the linker of wild-type protein G is 14 amino acid residues, which is only about 50 Å even considering the extended structure. If the number of structural domains contained in the protein is increased, it becomes sufficiently long, but when the number of structural domains increases, the structure of the protein becomes complicated, and therefore, during the culture process, for example, problems such as easy generation of precipitates in the solution are likely to occur, and further , as the number of domains increases and the overall size of the protein becomes larger, diffusion within the carrier becomes poorer.

In contrast, in the case of the protein of the present invention, the length of the maximum elongation of the linker between the bonding domains is adjusted in the range of about 80 Å to 240 Å, so even a protein containing two domains The dimer can also sterically and rationally divalently interact with the Fc region of the antibody, and there is no problem as described above.

In addition, examples of immunoglobulin G include human and non-human animals, especially various antibodies of mammals such as rats, mice, hamsters, goats, and rabbits, and various antibodies such as Fab fragments of human IgG. various fragments of . As long as it has an Fc portion or a Fab portion, its structure or constituent elements are not particularly limited, and any of various types of antibody molecules and fragment molecules thereof known to those skilled in the art are included. That is, in addition to ordinary (complete) IgG-type antibody molecules, for example, single-chain antibodies (scFv), dimers of single-chain antibodies, bispecific antibodies, diabody-type bispecific antibodies, and Multimerized low-molecular-weight antibodies, and various antibody fragments such as Fab fragment, F(ab') ₂ and Fab'.

Preferred embodiments of the above domain mutants included in the protein of the present invention include the following mutant proteins.

A. The first embodiment of the mutant protein of the present invention is shown in (1) and (2) below.

(1) Asp22, Ala24, Thr25, Lys28, Val29, Lys31, Gln32, Asn35, Asp36 in the wild-type protein G.B1 composed of the amino acid sequence shown in SEQ ID NO: 1 or 2 or the same B2 domain protein A mutant protein in which any one or more amino acid residues of Gly38, Asp40, Glu42, and Thr44 are substituted by other amino acid residues as the site to be mutated, wherein each amino acid residue at the site to be mutated is replaced by the following (i ) to (iii), the mutant protein has binding activity to the Fc region of immunoglobulin G, and compared with the wild-type protein G·B1 or B2 domain protein, The binding activity to the weakly acidic region of the Fc region of immunoglobulin G is reduced.

(i) When the amino acid residue at the site to be mutated is a non-charged amino acid residue, substitution to a charged amino acid residue

(ii) When the amino acid residue at the site to be mutated is a charged amino acid residue, substitution to a charged amino acid residue showing an opposite charge

(iii) Substitution of an amino acid residue at a site to be mutated into a histidine residue.

(2) one of Asp22, Thr25, Lys28, Lys31, Gln32, Asn35, Asp36, Gly38, Asp40, and Thr44 in the wild-type protein G.B3 domain protein composed of the amino acid sequence shown in SEQ ID NO: 3 A mutant protein in which any one or more amino acid residues are substituted by other amino acid residues as a site to be mutated, wherein each amino acid residue at the site to be mutated is represented by any one of the following (i) to (iii) Amino acid residue substitution, the mutant protein has binding activity to the Fc region of immunoglobulin G, and compared with the wild-type protein G·B3 domain protein, the weak acidic region of the Fc region of immunoglobulin G decreased binding activity.

(i) When the amino acid residue at the site to be mutated is a non-charged amino acid residue, substitution to a charged amino acid residue

(ii) When the amino acid residue at the site to be mutated is a charged amino acid residue, substitution to a charged amino acid residue showing an opposite charge

(iii) Substitution of an amino acid residue at a site to be mutated into a histidine residue.

The mutant proteins of (1) and (2) above are designed based on the site to be mutated and the amino acid residue substituted for the site selected as follows, and obtained by genetic engineering.

[Selection of sites to be mutated based on analysis of the binding surface to Fc and specification of amino acid residues to be substituted]

The site where the mutation was introduced for designing the amino acid sequence of the mutant protein of the present invention was selected using the atomic coordinate data of the three-dimensional structure of the complex bound to the protein G·B2 domain and the Fc region of immunoglobulin G (Reference 4). Certainly. In order to reduce the antibody-binding property of the extracellular domain of protein G in the weakly acidic region, the amino acid residues on the binding surface of the extracellular domain of protein G and the surrounding amino acid residues directly related to the binding to the Fc region The base can be substituted from wild type to non-wild type. Therefore, first, in a complex bound to the protein G·B2 domain and the Fc region of immunoglobulin G, the amino acid residue of the protein G·B2 domain present within a certain distance from the Fc region is specified, and the This serves as a candidate for a site to be mutated. Next, in order to minimize the structural instability of the extracellular domain of protein G accompanied by amino acid substitutions, among the above candidates, only the amino acid residues exposed on the molecular surface of the protein G·B2 domain were identified as mutations object part.

Therefore, specifically, as shown in Examples described later, by setting the above-mentioned distance range within 6.5 Å and setting the exposed surface area ratio at 40% or more, the wild-type amino acid sequence of the protein G·B2 domain ( Thirteen of Asp22, Ala24, Thr25, Lys28, Val29, Lys31, Gln32, Asn35, Asp36, Gly38, Asp40, Glu42, and Thr44 among SEQ ID NO: 2) were selected as mutation target sites.

In addition, as mentioned above, since the sequence identity of each cell extramembrane domain of protein G is extremely high, and there is almost no difference in the three-dimensional structure of the B1, B2, and B3 domains, insights into the three-dimensional structure of the B2 domain-Fc complex Also applicable to B1 and B3 domain-Fc complexes. Therefore, for the 13 sites to be mutated that are derived from the three-dimensional structure of the B2 domain-Fc complex, as long as there are amino acids of the same type at corresponding positions, not only in the B2 domain but also in the B1 and B2 domains Select the site to be mutated. That is, 13 of Asp22, Ala24, Thr25, Lys28, Val29, Lys31, Gln32, Asn35, Asp36, Gly38, Asp40, Glu42, and Thr44 in the wild-type amino acid sequence (SEQ ID NO: 1) of the protein G.B1 domain In addition, 10 of Asp22, Thr25, Lys28, Lys31, Gln32, Asn35, Asp36, Gly38, Asp40, and Thr44 among the wild-type amino acid sequence (SEQ ID NO: 3) of the protein G·B3 domain were selected is the mutation target site.

On the other hand, the amino acid residue to be substituted for the original amino acid residue at the site to be mutated is specified in any one of the following (i) to (iii).

(i) The wild-type amino acid residue at the site to be mutated has an uncharged side chain amino acid (Gly, Ala, Val, Leu, Ile, Ser, Thr, Asn, Gln, Phe, Tyr, Trp, Met, Cys , Pro), it is substituted with an amino acid (Asp, Glu, Lys, Arg, His) having a charged side chain. Charged amino acids vary greatly in chemical state depending on pH, so the antibody-binding property of protein G·B2 domain can be changed between neutral region and weakly acidic region.

(ii) When the wild-type amino acid residue at the site to be mutated is a charged amino acid, it is substituted with a charged amino acid showing an opposite charge. As described above, the chemical state of charged amino acids varies greatly depending on pH, so the antibody-binding property of the protein G·B2 domain can be changed between a neutral region and a weakly acidic region.

(iii) When the wild-type amino acid residue at the site to be mutated is other than histidine, it is substituted with histidine. The chemical state of histidine changes greatly in the neutral region and the weakly acidic region, so the antibody binding property of the protein G·B2 domain can be changed in the neutral region and the weakly acidic region.

Specifically, as shown in the examples below, as the amino acid residues at substitution positions, specifically specify Lys, Arg, or His for Asp22, and specifically specify Asp, Glu, Lys, Arg, or His, specify Asp, Glu, Lys, Arg, or His for Thr25, specify Asp, Glu, or His for Lys28, specify Asp, Glu, Lys, Arg for Val29 (only B1, B2 domains) , or His, specify Asp, Glu, or His for Lys31, specify Asp, Glu, Lys, Arg, or His for Gln32, specify Asp, Glu, Lys, Arg, or His for Asn35, specify Lys for Asp36 , Arg, or His, specify Asp, Glu, Lys, Arg, or His for Gly38, specify Lys, Arg, or His for Asp40, specify Lys, Arg, or His for Glu42 (only B1, B2 domains) , specifically specifying Asp, Glu, Lys, Arg, or His for Thr44. However, when Asn35 is substituted by Lys and/or Asp36 is substituted by Glu, the amino acid sequence other than the substitution position becomes identical to the amino acid sequence of each cell membrane domain of wild-type protein G by using the specified amino acid sequence of these amino acid residues. . Thus, it is distinguished from the mutant protein whose stability of the protein G cell membrane domain is improved in the application of the present inventors mentioned later.

B. A second embodiment of the mutant protein of the present invention is shown in (3) below.

(3) Mutant protein, characterized in that Lys10, Thr11, Lys13, Gly14 in the wild-type protein G B1, B2 or B3 domain protein composed of any one of the amino acid sequences shown in SEQ ID NO: 1 to 3 Any one or more amino acid residues among Glu15, Thr16, Thr17, Asn35, Asp36, and Gly38 are used as mutation target sites, and replaced with other types of amino acid residues except cysteine, which have no effect on immunoglobulin G The Fc region of has binding activity and has reduced binding to the Fab region of immunoglobulin G compared to the corresponding wild-type protein G·B1, B2 or B3 domain protein. The mutant protein of the above (3) is designed based on the site to be mutated and the amino acid residue substituted for the site selected as follows, and obtained by genetic engineering methods.

[Selection of sites to be mutated based on analysis of the binding surface to Fab and specification of amino acid residues to be substituted]

The sites to introduce mutations for designing the amino acid sequence of the mutant protein of the present invention were selected using the atomic coordinate data of the three-dimensional structure of the complex bound to the protein G·B3 domain and the Fab region of immunoglobulin G (Reference 5). Certainly. It is known that the extracellular domain of protein G also binds to the Fc region of immunoglobulin G and also binds to the Fab region (Reference 2). Therefore, one antibody molecule can bind to multiple extracellular domains of protein G at the same time, and in this state, the interaction between the antibody and the extracellular domains of protein G becomes multivalent, making it difficult to cleavage. Therefore, in order to reduce the antibody-binding property of the extracellular domain of protein G in the weakly acidic region, it is only necessary to replace the amino acid residues on the binding surface of the extracellular domain of protein G directly related to the binding to the Fab region from the wild type It is not a wild type. Therefore, first, in the complex bound to the protein G-B3 domain and the Fab region of immunoglobulin G, the amino acid residues of the protein G and each cell membrane B3 domain present within a certain distance from the Fab region are specified. , and use it as a candidate for the mutation target site. Next, in order to minimize the structural instability of the extracellular domain of protein G accompanied by amino acid substitutions, among the above candidates, only the amino acid residues exposed on the molecular surface of the protein G·B3 domain were identified as mutations object part.

Specifically, as shown in the examples described later, by setting the above-mentioned distance range within 4.0 Å and making the exposed surface area ratio 40% or more, the protein G·B3 structure shown in [SEQ ID NO: 3] Ten sites of Lys10, Thr11, Lys13, Gly14, Glu15, Thr16, Thr17, Asn35, Asp36, and Gly38 in the wild-type amino acid sequence of the domain were selected as sites to be mutated. In addition, as described above, the extracellular domains of protein G have extremely high identity as described above, and these mutation target sites also co-exist in any of the domains of B1, B2, and B3. Therefore, not only the B3 domain but also the B1 and B2 domains can be selected as mutation target sites.

On the other hand, the amino acid residues to be substituted for the original amino acid residues at the site to be mutated can be specified by the following method. (iv) Substitution with wild-type amino acid and other types of amino acid residues other than cysteine. Thus, in addition to avoiding the risk of cross-linking reactions associated with the introduction of cysteine, the mutation of the wild-type amino acid can lead to a decrease in binding to the Fab region.

Specifically, as shown in Examples described later, as amino acid residues to substitute for each extracellular domain protein of wild-type protein G, residues other than Lys and Cys were specified for Lys10, and Thr and Cys were specified for Thr11. Residues other than Lys and Cys are specified for Lys13, residues other than Gly and Cys are specified for Gly14, residues other than Glu and Cys are specified for Glu15, Thr16 is specified Thr Residues other than Thr and Cys are specified for Thr17, residues other than Asn and Cys are specified for Asn35, residues other than Asp and Cys are specified for Asp36, residues other than Asp and Cys are specified for Asp36, residues other than Asp and Cys are specified for Asp36, residues other than Asp and Cys are specified for Residues other than Gly and Cys are specified. However, when Asn35 is replaced by Lys and/or Asp36 is replaced by Glu by using the selected amino acid sequence of these amino acid residues, the amino acid sequence other than the substitution position becomes the same as the amino acid sequence of each cell membrane domain of wild-type protein G . Thus, it is distinguished from the mutant protein whose stability of the protein G cell membrane domain is improved in the application of the present inventors mentioned later.

C. The third embodiment of the mutant protein of the present invention shares amino acid residue substitutions for improving binding to the immunoglobulin Fc region and amino acid residue substitutions for improving binding to the Fab region.

[Selection of sites to be mutated based on analysis of binding surfaces to Fc and Fab and specification of amino acid residues to be substituted]

The site to be mutated and the amino acid residue to be substituted were combined by combining the site to be mutated and the amino acid residue to be substituted based on the analysis of the binding surface to Fab, and the site to be mutated. Specific Designation of Selected and Substituted Amino Acid Residues. Specifically, Asp22, Ala24, Thr25, Lys28, Val29, Lys31, Gln32, Asp40, Twenty sites of Glu42, Thr44, Lys10, Thr11, Lys13, Gly14, Glu15, Thr16, Thr17, Asn35, Asp36, and Gly38 were selected as mutation target sites. Among them, Asp22, Ala24, Thr25, Lys28, Val29, Lys31, Gln32, Asp40, Glu42, and Thr44 are target sites for improving binding to the Fc region, and Lys10, Thr11, Lys13, Gly14, Glu15, Thr16, and Thr17 are Target site for improved binding to the Fab region. Here, Asn35, Asp36, and Gly38 are sites for improving binding to the Fc region and also improving sites for binding to the Fab region. Therefore, the substitution of the amino acid residues shown in A above for improving the binding ability of Asn35, Asp36, and Gly38 to the Fc region is also simultaneously used for improving the binding ability to the Fab region except cysteine Substitutions to other amino acid residues other than residues.

That is, the "consensus" of amino acid residue substitutions referred to in this specification means that the site to be mutated for improving the binding property to the Fc region and the site to be mutated for improving the binding property to the Fab region are replaced by When selecting differently, except for the case of combining amino acid residue substitutions, the same site is selected as the site to be mutated for the improvement of the binding properties of both, and the amino acid residues shown in A above are selected. Substitution of groups is also defined in this sense.

As amino acid residues to be substituted, Lys, Arg, or His was selected for Asp22, Asp, Glu, Lys, Arg, or His was selected for Ala24, Asp, Glu, Lys, Arg, or His was selected for Thr25, and for Lys28 Select Asp, Glu, or His, select Asp, Glu, Lys, Arg, or His for Val29, select Asp, Glu, or His for Lys31, select Asp, Glu, Lys, Arg, or His for Gln32, Select Lys, Arg, or His for Asp40, select Lys, Arg, or His for Glu42, select Asp, Glu, Lys, Arg, or His for Thr44, select residues other than Lys and Cys for Lys10, Select residues other than Thr and Cys for Thr11, select residues other than Lys and Cys for Lys13, select residues other than Gly and Cys for Gly14, select residues other than Glu and Cys for Glu15 For Thr16 select residues other than Thr and Cys, for Thr17 select residues other than Thr and Cys, for Asn35 select residues other than Asn and Cys, for Asp36 select residues other than Asp and Cys Residues other than Gly and Cys were selected for Gly38.

On the other hand, for the protein G·B3 domain, Asp22, Thr25, Lys28, Lys31, Gln32, Asp40, Thr44, Lys10, Thr11, Lys13, Gly14, Seventeen sites including Glu15, Thr16, Thr17, Asn35, Asp36, and Gly38 were selected as mutation target sites. Among them, Asp22, Thr25, Lys28, Lys31, Gln32, Asp40, and Thr44 are mutation target sites for improving binding to the Fc region, and Lys10, Thr11, Lys13, Gly14, Glu15, Thr16, and Thr17 are for improving binding to the Fab region. Region binding mutation target site. Among them, Asn35, Asp36, and Gly38 are the improved sites for the binding to the Fc region and are also the improved sites for the binding to the Fab region. This point is common, and they are used for Asn35, Asp36, and Gly38 to the Fc region. The substitution of the amino acid residues shown in A above to improve the binding properties is also a substitution of other amino acid residues other than cysteine residues for improving the binding properties to the Fab region, which is different from the above-mentioned Protein G·B1 or B2 domains are the same.

However, the amino acid sequence set based on the above-mentioned selection of amino acid residues is the same as the amino acid sequence of each cell membrane domain of wild-type protein G except for the substitution of Asn35 with Lys and/or the substitution of Asp36 with Glu. same situation. Thus, it is distinguished from the mutant protein whose stability of the protein G cell membrane domain is improved in the application of the present inventors mentioned later.

Specific examples of the mutant protein of the present invention are shown in (a) to (c) below.

(a) is a mutant protein of the wild-type protein G.B1 domain protein, which consists of the amino acid sequence shown in (a) below, or consists of one or more amino acids in the amino acid sequence shown in (a) Amino acid sequence consisting of deletion, substitution, insertion or addition of residues, has binding activity to the Fc region of immunoglobulin G, and, compared with the wild-type protein G·B1 domain protein, at least, to the binding activity of immunoglobulin G The binding activity of the Fab region and/or the binding activity of the weakly acidic region to the Fc region is reduced.

(In the above amino acid sequences, X35 represents Asn or Lys, X36 represents Asp or Glu, X37 represents Asn, His, or Leu, X47 represents Asp or Pro, X48 represents Ala, Lys or Glu, X22 represents Asp or His, respectively, in the above amino acid sequences, X25 represents Thr or His, X32 represents Gln or His, X40 represents Asp or His, X42 represents Glu or His, X11 represents Thr or Arg, and X17 represents Thr or Ile. However, X35 is Asn or Lys and X36 is Asp simultaneously. or Glu, X37 is Asn or Leu, X47 is Asp or Pro, X48 is Ala, Lys or Glu, X22 is Asp, X25 is Thr, X32 is Gln, X40 is Asp, X42 is Glu, X11 is Thr, and X17 is In the case of Thr.).

(b) is a mutant protein of the wild-type protein G.B2 domain protein, which consists of the amino acid sequence shown in (b) below, or consists of one or more amino acids in the amino acid sequence shown in (b) Amino acid sequence consisting of deletion, substitution, insertion or addition of residues, has binding activity to the Fc region of immunoglobulin G, and, compared with the wild-type protein G·B2 domain protein, at least, to the binding activity of immunoglobulin G The binding activity of the Fab region and/or the binding activity of the weakly acidic region to the Fc region is reduced.

(In the above amino acid sequences, X35 represents Asn or Lys, X36 represents Asp or Glu, X37 represents Asn, His, or Leu, X47 represents Asp or Pro, X48 represents Ala, Lys or Glu, X22 represents Asp or His, respectively, in the above amino acid sequences, X25 represents Thr or His, X32 represents Gln or His, X40 represents Asp or His, X42 represents Glu or His, X11 represents Thr or Arg, and X17 represents Thr or Ile. However, X35 is Asn or Lys and X36 is Asp simultaneously. or Glu, X37 is Asn or Leu, X47 is Asp or Pro, X48 is Ala, Lys or Glu, X22 is Asp, X25 is Thr, X32 is Gln, X40 is Asp, X42 is Glu, X11 is Thr, and X17 is In the case of Thr.).

(c) is a mutant protein of the wild-type protein G.B3 domain protein, which consists of the amino acid sequence shown in (c) below, or consists of one or more amino acids in the amino acid sequence shown in (c) Amino acid sequence consisting of deletion, substitution, insertion or addition of residues, has binding activity to the Fc region of immunoglobulin G, and, compared with the wild-type protein G B3 domain protein, at least, to the binding activity of immunoglobulin G The binding activity of the Fab region and/or the binding activity of the weakly acidic region to the Fc region is reduced.

(in the above amino acid sequences, respectively, X35 represents Asn or Lys, X36 represents Asp or Glu, X37 represents Asn, His, or Leu, X47 represents Asp or Pro, X48 represents Ala, Lys or Glu, X22 represents Asp or His, X25 represents Thr or His, X32 represents Gln or His, X40 represents Asp or His, X11 represents Thr or Arg, and X17 represents Thr or Ile. However, X35 is Asn or Lys, X36 is Asp or Glu, and X37 is Asn simultaneously. or Leu, X47 is Asp or Pro, X48 is Ala, Lys or Glu, X22 is Asp, X25 is Thr, X32 is Gln, X40 is Asp, X42 is Glu, X11 is Thr, and X17 is Thr.).

It should be noted that, in the definitions of amino acid residues (a) to (c) above, the condition is that they are compatible with each cell membrane domain protein of wild-type protein G and the cell membrane-enhancing protein G involved in the application of the present inventors described later. The mutant proteins were distinguished by domain stability.

More specific mutant proteins of the present invention are shown in (d) to (i) below.

(d) is a mutant protein of the wild-type protein G.B1 domain protein, which consists of the amino acid sequence shown in (d) below, or consists of one or more amino acids in the amino acid sequence shown in (d) Composed of amino acid sequences with residue deletions, substitutions, insertions or additions, have binding activity to the Fc region of immunoglobulin G, and compared with wild-type protein G·B1 domain protein, have higher binding activity to the Fab region of immunoglobulin G Decreased binding activity and/or binding activity to Fc region in weakly acidic region.

(in the above amino acid sequences, respectively, X22 represents Asp or His, X25 represents Thr or His, X32 represents Gln or His, X40 represents Asp or His, X42 represents Glu or His, X11 represents Thr or Arg, X17 represents Thr or Ile However, the case where X22 is Asp, X25 is Thr, X32 is Gln, X40 is Asp, X42 is Glu, X11 is Thr, and X17 is Thr simultaneously is excluded.).

(e) is a mutant protein of the wild-type protein G.B2 domain protein, which consists of the amino acid sequence shown in (e) below, or consists of one or more amino acids in the amino acid sequence shown in (e) Composition of amino acid sequence with deletion, substitution, insertion or addition of residues, has binding activity to the Fc region of immunoglobulin G, and compared with wild-type protein G·B2 domain protein, has a higher binding activity to the Fab region of immunoglobulin G Decreased binding activity and/or binding activity to Fc region in weakly acidic region.

(in the above amino acid sequences, respectively, X22 represents Asp or His, X25 represents Thr or His, X32 represents Gln or His, X40 represents Asp or His, X42 represents Glu or His, X11 represents Thr or Arg, X17 represents Thr or Ile However, the case where X22 is Asp, X25 is Thr, X32 is Gln, X40 is Asp, X42 is Glu, X11 is Thr, and X17 is Thr simultaneously is excluded.).

(f) is a mutant protein of the wild-type protein G.B3 domain protein, which consists of the amino acid sequence shown in (f) below, or consists of one or more amino acids in the amino acid sequence shown in (f) The amino acid sequence composition of residue deletion, substitution, insertion or addition has binding activity to the Fc region of immunoglobulin G, and compared with the wild-type protein G·B3 domain protein, it has a higher binding activity to the Fab region of immunoglobulin G Decreased binding activity and/or binding activity to Fc region in weakly acidic region.

(In the above amino acid sequences, X22 represents Asp or His, X25 represents Thr or His, X32 represents Gln or His, X40 represents Asp or His, X11 represents Thr or Arg, X17 represents Thr or Ile, respectively. However, simultaneously X22 is Asp, X25 is Thr, X32 is Gln, X40 is Asp, X11 is Thr, and X17 is Thr.).

(g) is a mutant protein of the wild-type protein G.B1 domain protein, which consists of the amino acid sequence shown in (g) below, or consists of one or more amino acids in the amino acid sequence shown in (g) Amino acid sequence composed of deletion, substitution, insertion or addition of residues, has binding activity to the Fc region of immunoglobulin G, and, compared with the wild-type protein G·B1 domain protein, has a weak acidic effect on the Fc region Reduced binding activity.

(In the above amino acid sequences, X22 represents Asp or His, X25 represents Thr or His, X32 represents Gln or His, X40 represents Asp or His, X42 represents Glu or His, respectively. However, X22 is Asp and X25 is The case where Thr, X32 is Gln, X40 is Asp, and X42 is Glu.).

(h) is each mutant protein of the wild-type protein G·B2 domain protein, which consists of the amino acid sequence shown in (h) below, or consists of one or more of the amino acid sequences shown in (h) Amino acid residue deletion, substitution, insertion or addition amino acid sequence composition, has the binding activity to the Fc region of immunoglobulin G, and compared with the wild-type protein G·B2 domain protein, it has a weak acidic effect on the Fc region Reduced binding activity.

(In the above amino acid sequences, X22 represents Asp or His, X25 represents Thr or His, X32 represents Gln or His, X40 represents Asp or His, X42 represents Glu or His, respectively. However, X22 is Asp and X25 is The case where Thr, X32 is Gln, X40 is Asp, and X42 is Glu.).

(i) is each mutant protein of the wild-type protein G·B3 domain protein, which consists of the amino acid sequence shown in (i) below, or consists of one or more of the amino acid sequences shown in (i) Amino acid residue deletion, substitution, insertion or addition amino acid sequence composition, has the binding activity to the Fc region of immunoglobulin G, and compared with the wild-type protein G·B3 domain protein, it has a weak acidic effect on the Fc region Reduced binding activity.

(In the above amino acid sequences, X22 represents Asp or His, X25 represents Thr or His, X32 represents Gln or His, and X40 represents Asp or His, respectively. However, simultaneously X22 is Asp, X25 is Thr, X32 is Gln, and X40 is the case for Asp). It should be noted that, in the definitions of the amino acid residues in (d) to (i) above, the condition is that they are different from each cell membrane domain protein of wild-type protein G.

As can be seen from the above, in the design of the mutant protein of the present invention, the selected mutation target site and the amino acid residue to replace the site are not limited to one each, and therefore can be selected from among the mutation target site and the amino acid residue to replace the site. Appropriately selected, the amino acid sequence of the mutant protein is designed. For example, in order to improve the binding property of the Fc region of immunoglobulin G, Asp22, Thr25, Gln32, Asp40, and Glu42 in the amino acid sequence of the wild-type protein G·B1 or B2 domain are selected as the site to be mutated. For the corresponding substituted amino acid residues, select Asp22His, Thr25His, Gln32His, Asp40His, and Glu42His, and perform any one amino acid on the wild-type amino acid sequence (SEQ ID NO: 1, 2) of the protein G·B1 or B2 domain Amino acid sequences of multiple mutant proteins can be designed by substituting or combining point mutations or multiple mutations of up to 5 mutation positions/5 substitutions of these amino acid substitutions. The amino acid sequences of (g) and (h) above show such point mutations and multiple mutations up to a maximum of 5 mutation positions/5 substitutions, and are examples of mutant proteins of the present invention.

In addition, examples of improved mutant proteins that further increase binding to the Fab region in addition to the above-mentioned improvement of binding to the Fc region of immunoglobulin G include, for example, protein G, B1 or B2 The wild-type amino acid sequence of the structural domain is a mutated mutant protein with a maximum of 7 mutation positions/7 substitutions, wherein Thr11 and Thr17 of the wild-type protein G·B1 or B2 domain are selected as the amino acid residues corresponding to the substitution , select Thr11Arg and Thr17Ile, and add them to the above-mentioned mutations up to a maximum of 5 mutation positions/5 substitutions to introduce a maximum of 7 mutation positions/7 substitutions. The amino acid sequences of (d) and (e) above show examples of point mutations or multiple mutations up to a maximum of 7 mutation positions/7 substitutions. Among these amino acid sequences, there are Thr11Arg and/or Thr17Ile mutations, and there are The amino acid sequence of any one or more mutations among the mutations shown above Asp22His, Thr25His, Gln32His, Asp40His, and Glu42His further improves the binding to the Fab region in addition to improving the binding to the Fc region of immunoglobulin G sex.

On the other hand, the amino acid sequence of (i) above is an example of the amino acid sequence of a mutant protein of the wild-type protein G·B3, except for Asp22His, Thr25His, and Gln32His, which are the above-mentioned mutations for improving binding to the Fc region. The amino acid sequences of (g) and (h) were designed in the same manner as the amino acid sequences of (g) and (h) except any one or more mutations in Asp40His, up to a maximum of 4 mutation positions/4 substitutions. In addition, the amino acid sequence of the above (f) is an example of the amino acid sequence of the mutant protein of the wild-type protein G·B3, except for Asp22His, Thr25His, Gln32His, Asp40His, which are the above-mentioned mutations for improving binding to the Fc region. In addition to any one or more of Thr11Arg and Thr17Ile mutations for improving binding to the Fab region, up to a maximum of 6 mutation positions/6 substitutions, and (d) and (e) Amino acid sequences were similarly designed.

In the present invention, in addition to such mutations, mutations known to optimize the properties of the extracellular domain of protein G can be further added. According to the previous studies of the present inventors, for example, there are mutation methods for improving the thermal stability of the extracellular domain of protein G, the chemical stability to denaturants, and the resistance to decomposing enzymes (Patent Document 6). That is, introduction of one or more mutations among Asn35Lys, Asp36Glu, Asn37His, Asn37Leu, Asp47Pro, Ala48Lys, and Ala48Glu improves the above-mentioned stability of the protein G·B1, B2, or B3 domain. These mutations can make the mutant protein of the present invention more useful by combining the above-mentioned mutations for improving the binding properties to the Fc region of immunoglobulin G and/or the binding properties to the Fab region in the present invention.

For example, by adding these mutations to the above-mentioned maximum 7 mutation positions/7 substitutions to the wild-type amino acid sequence (SEQ ID NO: 1, 2) of the protein G·B1 or B2 domain, up to a maximum of 12 Mutation position/multiple mutations of 14 substitutions can design amino acid sequences of multiple mutant proteins that are further stabilized.

The amino acid sequences of (a) and (b) above show such point mutations and multiple mutations with a maximum of 12 mutation positions/14 substitutions, but only stabilizing mutations added to the wild-type sequence are excluded.

In the amino acid sequences of (a) and (b), the introduction of any one or more mutations among Asn35Lys, Asp36Glu, Asn37His, Asn37Leu, Asp47Pro, Ala48Lys, and Ala48Glu, except for the above-mentioned maximum 7 mutation positions/7 substitutions In addition to the improvement of the binding properties of the Fab region of immunoglobulin G and/or the improvement of the binding properties of the Fc region in the weakly acidic region, it also improves the stability of the protein G·B1 or B2 domain mutant protein.

On the other hand, the above (c) shows an example of the amino acid sequence of a mutant protein of the wild-type protein G·B3, showing point mutations and multiple substitutions with a maximum of 11 mutation positions/13 substitutions, but in addition to being the above-mentioned Fc The amino acid sequences of (a) and (b) are the same as the amino acid sequences of (a) and (b) except any one or more of mutations for improving binding properties of the region, up to a maximum of 4 mutation positions/4 substitutions design. Therefore, in the amino acid sequence of (c), introduction of any one or more mutations among Asn35Lys, Asp36Glu, Asn37His, Asn37Leu, Asp47Pro, Ala48Lys, and Ala48Glu, in addition to the binding properties to the Fab region of immunoglobulin G and/or to In addition to the improvement of the binding properties of the Fc region in the weakly acidic region, the stability of the protein G·B3 domain mutant protein is also improved.

The site to be mutated in the present invention is selected using the atomic coordinate data of the three-dimensional structure of the B2 domain-Fc complex and the B3 domain-Fab complex of protein G as described above, but the B1 domain is not only in the amino acid The sequence (Fig. 2) and the three-dimensional structure have almost no difference with the B2 domain, so the effects of the above-mentioned selected mutations are equally effective for the respective domains. In addition, the B3 domain hardly differs from the B2 domain not only in the amino acid sequence ( FIG. 2 ) but also in the three-dimensional structure, so the effects of the above-mentioned selected mutations in the B2 domain are equally effective for each domain.

For example, the wild-type amino acid residues at the above-mentioned selected 12 mutation positions are all common between the above-mentioned B2 domain and the above-mentioned B1 domain. Therefore, a combination of the above-mentioned selected 5 mutation positions/5 substitutions, or 7 mutation positions/7 substitutions, or 12 mutation positions/14 substitutions is introduced into the B1 amino acid sequence with high identity with the B2 domain. Point mutations and multiple mutations can be used as amino acid sequences of mutant proteins of the B1 domain. In addition, the wild-type amino acids at the above-mentioned 12 selected mutation positions are all common between the above-mentioned B2 domain and the above-mentioned B3 domain as long as position 42 is excluded (the wild-type amino acid residue at position 42 in the B2 domain is Glu42, In the B3 domain is Val42). Therefore, for the amino acid sequence of the B3 domain with high identity with the B2 domain, the introduction will be selected from the above-mentioned 5 mutation positions/5 substitutions, or 7 mutation positions/7 substitutions, or 12 mutation positions/ 14 substitutions 4 mutation positions/4 substitutions, or 6 mutation positions/6 substitutions, or 11 mutation positions/13 substitutions combined point mutations and multiple mutations except the mutation position at position 42, can be used as B3 Domain amino acid sequence of the mutant protein. As shown in the examples described later, mutants of the B1 domain can also be selected from the atomic coordinate data of the three-dimensional structures of the B2 domain-Fc complex and the B3 domain-Fab complex of protein G. Proteins are known to have desirable properties.

As mentioned above, the amino acid sequence of the mutant protein of the present invention is not limited to one, and there are several. Among them, when specific examples of preferred sequences are given, for example, [SEQ ID NO: 13], [SEQ ID NO: 14 ], [SEQ ID NO:15], [SEQ ID NO: 16], [SEQ ID NO: 17], [SEQ ID NO: 18], [SEQ ID NO: 19], or [SEQ ID NO: 20] The amino acid sequence shown.

The mutant protein shown in [SEQ ID NO: 13] is a wild-type amino acid sequence of the G·B1 domain of the protein shown in [SEQ ID NO: 1], which is improved from the previous research of the inventors. A protein that introduces mutations into the thermal stability of the extracellular domain of protein G, the chemical stability of denaturing agents, and the resistance to decomposing enzymes, [SEQ ID NO: 14], [SEQ ID NO: 15], The mutant proteins shown in [SEQ ID NO: 19] and [SEQ ID NO: 20] are, in addition, proteins in which mutations have been introduced into a site selected based on analysis of the binding surface to Fc.

On the other hand, the mutant proteins shown in [SEQ ID NO: 16], [SEQ ID NO: 17], and [SEQ ID NO: 18] are protein G shown in [SEQ ID NO: 1]. The wild-type amino acid sequence of the B1 domain is based on the previous studies of the inventors, and it is known that the thermal stability of the extracellular domain of protein G, the chemical stability to denaturing agents, and the resistance to decomposing enzymes are improved. , and Fab-based binding surface analysis of selected sites to introduce mutant proteins.

As long as the mutant protein of the present invention has binding activity to antibodies or immunoglobulin G or a protein having an Fc region of immunoglobulin G, compared with each protein of the extracellular membrane domain of wild-type protein G, at least, to immunoglobulin G If the binding activity of the Fab region of protein G and/or the binding activity to the Fc region in the weakly acidic region is reduced, then in any one of the amino acid sequences shown in the above-mentioned mutant protein of the present invention, one or more (for example, 2 2 to 5) of amino acid residues may undergo mutations such as deletion, substitution, insertion, addition, etc., so that their sequence identity with each of the reference amino acid sequences is 90% or more, preferably 95% or more, more preferably More than 98%.

Furthermore, examples of domain mutants contained in proteins used in the following examples include polypeptides having the following specific amino acid sequence [SEQ ID NO: 19].

(j) AspThrTyrLysLeuIleLeuAsnGlyLysThrLeuLysGlyGluThrThrGluThrAlaValAspAlaAlaThrAlaGluLysValPheLysHisTyrAlaAsnGluHisGlyValHisGlyHisTrpThrTyrAspProGluThrLysThrPheThrValThrGlu.

In addition, the protein of the present invention may also be a fusion protein composed of a fusion-type amino acid sequence linked to an amino acid sequence of any other protein at the N-terminal or C-terminal side. For example, it can also be used as [amino acid sequence (a)]-linker sequence E-protein A, or protein B-linker sequence F-[amino acid sequence (a)]-linker sequence G-protein C-linker sequence H-[amino acid sequence (c)]. Examples of other amino acid sequences used in such fusion proteins include the amino acid sequence of the oxaloacetate decarboxylase α-subunit c-terminal domain (OXADac) shown in [SEQ ID NO: 31]. At this time, the OXADac-protein G mutant fusion protein can have multiple functions of avidin-binding activity derived from the OXADac region and antibody-binding activity derived from the protein G mutant region in a single molecule.

For example, when the protein of the present invention is synthesized in the form of a fusion protein with a His tag attached or with another protein, sequence-specific proteolysis is performed between the tag and the mutant protein, or between the other protein and the protein of the present invention after synthesis. Enzymatic decomposition may leave one or more amino acid residues on the N-terminal side or C-terminal side of the protein of the present invention. In addition, during the production of the protein of the present invention using Escherichia coli, etc., it may be added to the N-terminal side. The activity of the protein of the present invention does not change by adding these amino acid residues, such as methionine derived from the initiation codon, as shown below. In addition, the effect of the designed mutation will not be lost by adding these amino acid residues. Therefore, the protein of the present invention will of course also contain these mutations. It should be noted that, in order to obtain the protein of the present invention without the addition of such amino acid residues, for example, the protein produced by E. The terminal amino acid residue (Reference 7) is obtained by separating and purifying the reaction mixture by chromatography or the like.

In this specification, in addition to the protein formed by bonding a plurality of domains having affinity for a protein having the Fc part of immunoglobulin G (IgG) with a linker, the above fusion protein is also included, and is also referred to simply as " protein". The present invention further relates to a nucleic acid encoding a related protein, a recombinant vector containing the nucleic acid, and a transformant introduced with the recombinant vector.

In addition, it also relates to a protein characterized in that the above-mentioned protein is immobilized on a water-insoluble solid-phase support, immunoglobulin G or a protein having an Fc region or a Fab region of immunoglobulin G (also referred to as "immunoglobulin G, etc.") A capturing agent for capturing agents, antibodies containing the capturing agents, affinity chromatography for purification of immunoglobulin G, etc., a method for purifying immunoglobulin G, etc., using the affinity chromatography for purification, and the like. Here, "having affinity" means, for example, that immunoglobulin G and the like can be adsorbed by chromatography. As shown in the following examples, by using the protein of the present invention, certain immunoglobulin G can be sufficiently adsorbed, for example, the Fab fragment of human IgG and rat IgG, therefore, the above-mentioned capture agent is particularly suitable as a capture agent for its purification Very useful.

In the purification method of the present invention, in order to utilize the above-mentioned protein as an antibody complementing agent, it is preferable to use an affinity column in which a glass tube or other column is filled with a filler in which the protein is immobilized in water typified by agarose beads. Insoluble carrier (water-insoluble solid phase support).

As the adsorption buffer, any buffer may be used as long as the pH is near neutral and the salt used can adjust the pH. Typically, a buffer in which an electrolyte such as sodium chloride is dissolved in a phosphate buffer or a Tris buffer is used. liquid. As the pH of the adsorption buffer, a buffer having a pH of 9.0 to 6.5, preferably a pH of 8.0 to 7.0 is used. In addition, as the elution buffer, it is only necessary to use a pH range of 6.5 to 2.0 as long as it is in the pH range where the target immunoglobulin G or the like is eluted. The type of elution buffer may be any known to those skilled in the art, and typical examples include phosphate buffer, citrate buffer, acetate buffer, glycine buffer, and the like.

The operation itself in the purification method of the present invention can be carried out by common operations known to those skilled in the art. That is, as in usual affinity purification, first, a sample solution containing immunoglobulin G and the like to be purified is injected into a column stabilized with an adsorption buffer, and immunoglobulin G and the like are adsorbed to the filler. Then, after washing away non-adsorbed components remaining in the column with an adsorption buffer, the adsorbed immunoglobulin G and the like are eluted with an elution buffer, and the immunoglobulin G and the like are recovered in the eluent. It should be noted that other affinity purification conditions such as the flow rate (flow rate) of the adsorption buffer and the elution buffer and column temperature can be appropriately determined by those skilled in the art.

As long as the sample solution is a substance containing immunoglobulin G, etc., such as serum or ascitic fluid culture solution, there are no restrictions on its source and other components. Furthermore, in the purification method of the present invention, as the purification means, it is only necessary to use the affinity between a protein containing a domain mutant (artificially mutated domain) and immunoglobulin G, etc., and in addition to the method using a column, Any means known to those skilled in the art, such as immunoprecipitation and immobilization of the protein to magnetic beads, can be used.

Preferable examples of mutants of the wild-type protein G·B1 domain contained in the protein of the present invention include mutant proteins described in Patent Document 8 above. Such a mutant protein can be easily prepared by, for example, the following method, as long as those skilled in the art are skilled in the art according to the method described in Patent Document 7 or Patent Document 8.

1. Protein Preparation

(1) Preparation of proteins using genetic engineering methods

a. Gene encoding the mutant protein

In the present invention, genetic engineering methods can be used in order to prepare the above-mentioned designed proteins.

The gene used in such a method is composed of nucleic acid encoding the following: the protein shown in the above-mentioned A~C, more specifically, the amino acid sequence shown in any one of the above-mentioned (a)~(i); or A protein having a deleted, substituted or added amino acid sequence at one or more amino acid residues in the amino acid sequence shown in any one of (a) to (i), which is compatible with an antibody or immunoglobulin G or has an immunoglobulin G Proteins in the Fc region have binding activity, and the binding activity is reduced in the weakly acidic region compared to the neutral region; for example, more specifically, those shown by [SEQ ID NO: 22] to [SEQ ID NO: 29] Nucleic acid consisting of either base sequence.

In addition, as the gene used in the present invention, a nucleic acid that hybridizes under stringent conditions to a nucleic acid composed of a sequence complementary to the base sequence of the above nucleic acid, which encodes an antibody or immunoglobulin G or has an immune The protein of the Fc region of globulin G has binding activity, and the binding activity to the Fab region of immunoglobulin G and/or the Fc region is in a weakly acidic region compared with the corresponding wild-type protein G and extracellular domain protein The aforementioned mutant proteins with reduced binding activity. Here, stringent conditions refer to conditions under which specific hybrids are formed and non-specific hybrids are not formed. For example, it refers to conditions under which nucleic acids with high identity (identity of 60% or more, preferably 80% or more, more preferably 90% or more, most preferably 90% or more) hybridize. More specifically, it refers to conditions where the sodium concentration is 150 to 900 mM, preferably 600 to 900 mM, and the temperature is 60 to 68°C, preferably 65°C. For example, when the hybridization condition is 65°C, the washing condition is 0.1×SSC containing 0.1% SDS at 65°C for 10 minutes, and the hybridization is confirmed by conventional methods such as Southern blotting, dot blot hybridization, etc., it is called to hybridize under stringent conditions.

The gene encoding the protein of the present invention includes the above nucleic acid and a nucleic acid encoding any linker sequence described above, depending on the desired structure of the protein of the present invention. Nucleic acids encoding each mutant protein constituting the tandem multimer and nucleic acids encoding a linker sequence may be alternately linked in multiples, or the nucleic acid may be linked with a nucleic acid encoding an amino acid sequence of an arbitrary protein to design a coding sequence. Fusion amino acid sequence.

b. Genes, recombinant vectors and transformants

The above-mentioned gene of the present invention can be synthesized by chemical synthesis, PCR, cassette mutagenesis, site-specific mutation introduction, or the like. For example, by chemically synthesizing a plurality of oligonucleotides having a complementary region of about 20 base pairs at the end to about 100 bases, and subjecting these combinations to the overlap extension method (Reference 8), the entire target gene can be synthesized .

The recombinant vector of the present invention can be obtained by linking (inserting) a gene containing the above nucleotide sequence into an appropriate vector. The vector used in the present invention is not particularly limited as long as it can replicate in the host or can integrate the target gene into the host genome. For example, bacteriophages, plasmids, cosmids, phagemids and the like can be mentioned.

Examples of plasmid DNA include plasmids derived from actinomycetes (such as pK4, pRK401, pRF31, etc.), plasmids derived from Escherichia coli (such as pBR322, pBR325, pUC118, pUC119, pUC18, etc.), plasmids derived from Bacillus subtilis (such as pUB110, pTP5, etc.), yeast-derived plasmids (eg, YEp13, YEp24, YCp50, etc.), and phage DNAs include λ phages (λgt10, λgt11, λZAP, etc.). Furthermore, animal viruses such as retrovirus and vaccinia virus, and insect virus vectors such as baculovirus can also be used.

To insert a gene into a vector, first, a method of cleaving purified DNA with an appropriate restriction enzyme, inserting an appropriate restriction enzyme site or a multiple cloning site of the vector DNA, and ligating it into the vector is used. It is necessary for the gene to be integrated into the vector in such a manner as to express the mutant protein of the present invention. Here, in the vector of the present invention, in addition to the promoter and the base sequence of the gene, cis-elements such as enhancers, splicing signals, poly A addition signals, selectable markers, ribosome binding sequences ( SD sequence), start codon, stop codon, etc. In addition, a tag sequence for purification of a protein that can be easily prepared may also be linked. As the tag sequence, base sequences encoding known tags such as His tag, GST tag, MBP tag, and BioEase tag can be used.

Whether or not the gene is inserted into the vector can be confirmed by known genetic engineering techniques. For example, in the case of a plasmid vector or the like, it can be confirmed by subcloning the vector using competent cells, extracting the DNA, and determining its base sequence using a DNA sequencer. For other vectors, bacteria or other hosts can also be used for subcloning, and the same method can be used. In addition, vector selection using selection markers such as drug resistance genes is also effective.

A transformant can be obtained by introducing the recombinant vector of the present invention into a host cell in such a manner that the mutant protein of the present invention can be expressed. The host used for transformation is not particularly limited as long as the protein or polypeptide can be expressed. Examples thereof include bacteria (Escherichia coli, Bacillus subtilis, etc.), yeast, plant cells, animal cells (COS cells, CHO cells, etc.), and insect cells.

When bacteria are used as the host, the recombinant vector is preferably capable of autonomous replication in the bacteria, and is composed of a promoter, a ribosome binding sequence, an initiation codon, a nucleic acid encoding the mutant protein of the present invention, and a transcription termination sequence. Examples of Escherichia coli include Escherichia coli BL21 and the like, and examples of Bacillus subtilis include Bacillus subtilis. The method for introducing a recombinant vector into bacteria is not particularly limited as long as it is a method of introducing DNA into bacteria. For example, heat shock method, method using calcium ions, electroporation method, etc. are mentioned.

When yeast is used as a host, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe and the like are used. The method for introducing a recombinant vector into yeast is not particularly limited as long as it is a method for introducing DNA into yeast, and examples thereof include electroporation, spheroplast method, and lithium acetate method.

When animal cells are used as hosts, monkey cells COS-7, Vero, Chinese hamster ovary cells (CHO cells), mouse L cells, rat GH3, human FL cells, etc. are used. Examples of methods for introducing recombinant vectors into animal cells include electroporation, calcium phosphate method, lipofection and the like.

When insect cells are used as hosts, Sf9 cells and the like are used. Examples of methods for introducing recombinant vectors into insect cells include calcium phosphate method, lipofection method, electroporation method and the like.

Whether or not the gene has been introduced into the host can be confirmed by PCR, DNA hybridization, RNA hybridization, or the like. For example, DNA is prepared from transformants and DNA-specific primers are designed for PCR. Next, the PCR amplification product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, or capillary electrophoresis, etc., stained with ethidium bromide, SyberGreen solution, etc., and the amplification product is detected as one band, Conversions can be confirmed. In addition, PCR may be performed using primers previously labeled with a fluorescent dye or the like to detect the amplified product.

c. Acquisition of proteins cultured using transformants

When produced as a recombinant protein, the protein of the present invention can be obtained by culturing the above-mentioned transformant and collecting it from the culture. The culture means any of culture supernatant, cultured cells, cultured cells, or crushed products of cells or cells. The method for culturing the transformant of the present invention is carried out according to the usual method used for culturing the host.

The medium for cultivating transformants obtained by using microorganisms such as Escherichia coli and yeast as hosts contains carbon sources, nitrogen sources, inorganic salts, etc. that can be used by microorganisms, and as long as it is a medium that can effectively cultivate transformants, it can be used. Any of natural medium and synthetic medium. Examples of the carbon source include carbohydrates such as glucose, fructose, sucrose, and starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol. Examples of nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, and other inorganic acids or ammonium salts of organic acids, or other nitrogen-containing compounds, peptone, meat extract, corn steep liquor, etc. . Examples of inorganic substances include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate. The culture is usually carried out at 20 to 37° C. for 12 hours to 3 days under aerobic conditions such as shaking culture or aeration and stirring culture.

After culturing, when the protein of the present invention is produced in cells or cells, the cells or cells are disrupted by repeated ultrasonic treatment, freezing and thawing, homogenizer treatment, etc. to collect the protein. In addition, when the protein is produced outside the cells or cells, the culture solution is used as it is, or the cells or cells are removed by centrifugation or the like. Thereafter, it can be separated and purified from the above-mentioned culture by using general biochemical methods used in the separation and purification of proteins, such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc., alone or in appropriate combination. Proteins of the invention.

In addition, when using a so-called cell-free synthesis system in which only factors (enzyme, nucleic acid, ATP, amino acid, etc.) (Reference 9). Thereafter, the mutant protein of the present invention can be isolated and purified from the reaction mixed solution using the same purification method as above.

In order to confirm whether the isolated and purified protein of the present invention is a protein consisting of the target amino acid sequence, a sample containing the protein is analyzed. As an analysis method, SDS-PAGE, Western blotting, mass spectrometry, amino acid analysis, amino acid sequencer, etc. can be used (Reference 10).

(2) Preparation of protein using other methods

The proteins of the present invention can also be prepared using organic chemical methods such as solid phase peptide synthesis and the like. Protein production methods using such methods are well known in the art and will be briefly described below.

When the protein is chemically prepared by solid-phase peptide synthesis, it is preferable to use an automatic synthesizer to synthesize a protected polypeptide having the amino acid sequence of the protein of the present invention on a resin by repeated polycondensation reactions of activated amino acid derivatives. Next, when the protected polypeptide is cut off from the resin, the protecting group of the side chain is also cut off at the same time. It is known that in this cleavage reaction, an appropriate mixture exists depending on the type of resin, protecting group, and amino acid composition (Reference 11). Thereafter, the crudely purified protein is transferred from the organic solvent layer to the aqueous layer to purify the target protein. As a purification method, reverse phase chromatography or the like can be used (Reference Document 11).

2. Protein immobilization

The protein used in the purification method of the present invention is utilized as a capture agent such as an antibody by utilizing its antibody-binding property.

The antibody capture agent of the present invention may be in any form as long as it contains the protein of the present invention. Preferably, the form in which the mutant protein of the present invention is immobilized on a water-insoluble solid support is suitable. Examples of water-insoluble carriers used include inorganic carriers such as glass beads and silica gel, synthetic polymers such as cross-linked polyvinyl alcohol, cross-linked polyacrylate, cross-linked polyacrylamide, and cross-linked polystyrene; Organic carriers made of polysaccharides such as natural cellulose, cross-linked cellulose, cross-linked agarose, cross-linked dextran, etc., and organic-organic, organic-inorganic composite carriers obtained by combining these, etc. Among them, the hydrophilic carrier has less non-specific adsorption, and the selectivity of antibodies, immunoglobulin G, or proteins having an Fc region of immunoglobulin G is good, so it is preferable. The hydrophilic carrier referred to here refers to a carrier whose contact angle with water is 60 degrees or less when the compound constituting the carrier is formed into a flat plate. Examples of such carriers include polysaccharides such as cellulose, chitosan, and dextran, polyvinyl alcohol, saponified ethylene-vinyl acetate copolymer, polyacrylamide, polyacrylic acid, and polymethacrylic acid. , polymethyl methacrylate, polyacrylic acid-grafted polyethylene, polyacrylamide-grafted polyethylene, glass, etc. are representative examples.

Examples of commercially available products include GCL2000 and GC700, which are porous cellulose gels, Sephacryl S-1000, which is covalently bonded to allyl dextran and methylenebisacrylamide, and Sephacryl S-1000, which is an acrylic carrier. Toyopearl, Sepharose CL4B as an agarose-based cross-linking carrier, Eupergit C250L as polymethacrylamide activated by epoxy groups, etc. However, the present invention is not limited to these carriers and activated carriers. The above-mentioned carriers may be used alone, or any two or more of them may be mixed. In addition, as the water-insoluble carrier used in the present invention, from the purpose and method of use of the antibody capture agent, it is desirable to have a large surface area, and it is preferable to have a large number of pores of an appropriate size, that is, to be porous.

As the form of the carrier, bead form, fiber form, film form (including hollow fiber) and the like are all possible, and any form can be selected. From the viewpoint of easiness of preparation of a carrier having a specific exclusion limit molecular weight, it is particularly preferable to use a bead shape. As for the average particle diameter of beads, an average particle diameter of 10 to 2500 µm is easy to use, and in particular, a range of 25 µm to 800 µm is preferable from the viewpoint of easiness of the ligand immobilization reaction.

Furthermore, when a functional group used in the immobilization reaction of the ligand exists on the surface of the carrier, it is suitable for immobilization of the ligand. Typical examples of these functional groups include hydroxyl group, amino group, aldehyde group, carboxyl group, mercapto group, silanol group, amide group, epoxy group, succinimide group, acid anhydride group, iodoacetyl group and the like.

In the immobilization of the mutant protein to the above-mentioned carrier, the capture efficiency is improved by reducing the steric hindrance of the mutant protein, and furthermore, in order to suppress non-specific binding, it is more preferable to immobilize through a hydrophilic spacer. . As the hydrophilic spacer, for example, it is preferable to use a polyalkylene oxide derivative in which both ends are substituted with carboxyl groups, amino groups, aldehyde groups, epoxy groups, or the like.

The method and conditions for immobilizing the mutant protein introduced into the above-mentioned carrier and the organic compound used as a spacer are not particularly limited, and generally, methods used for immobilizing proteins and peptides on the carrier are exemplified. Examples include activating the carrier by reacting it with cyanogen bromide, epichlorohydrin, diglycidyl ether, tosyl chloride, trifluoroethanesulfonyl chloride, hydrazine, etc. The reactive functional group of the compound immobilized by the ligand), the reaction with the compound immobilized as the ligand, the method of immobilization, in addition, by adding such as carbodiimide to the carrier and the system in which the compound immobilized as the ligand exists Condensation reagents or reagents with multiple functional groups in the molecule such as glutaraldehyde are condensed and cross-linked. However, it is suitable for the immobilization of proteins that are not easily detached from the carrier when the capture agent is sterilized or used. method is more preferable.

3. Performance confirmation test of protein and antibody capture reagents

The mutant protein and protein (hereinafter also referred to simply as "protein") prepared as above, and the antibody capture agent can be subjected to the following performance confirmation test to select a good material, and the protein and antibody capture material of the present invention have good performance. .

(1) Antibody binding test

The antibody-binding property of the protein of the present invention can be confirmed and evaluated by Western blotting, immunoprecipitation, pull-down assay, ELISA (enzyme-linked immunosorbent assay), surface plasmon resonance (SPR) and the like. Among them, the SPR method can quantitatively evaluate the binding reaction of mutant proteins from the viewpoint of velocity theory because the interaction between living bodies can be observed in real time and over time without labeling.

In addition, the antibody-binding property of the mutant protein immobilized on a water-insoluble solid support can be confirmed and evaluated by the above-mentioned SPR method and liquid chromatography. Among them, liquid chromatography can reliably evaluate the pH dependence of antibody binding properties.

(2) Thermal stability test of protein

The thermal stability of the mutant protein of the present invention can be evaluated by circular dichroism (CD) spectroscopy, fluorescence spectroscopy, infrared spectroscopy, differential scanning calorimetry, residual activity after heating, and the like. Among them, since CD spectroscopy is a spectroscopic analysis method that can sensitively reflect changes in the secondary structure of proteins, it is possible to quantitatively evaluate the structural stability by observing the changes in the three-dimensional structure of mutant proteins with respect to temperature.

〔references〕

Reference 1; Bjorck L, Kronvall G. (1984) Purification and some properties of streptococcal protein G, a novel IgG-binding reagent. J Immunol. 133, 69-74.

Reference 2; Boyle M. D.P., Ed. (1990) Bacterial Immunoglobulin Binding Proteins. Academic Press, Inc., San Diego, CA.

Reference 3; Gallagher T, Alexander P, Bryan P, Gilliland GL. (1994) Twocrystal structures of the B1 immunoglobulin-binding domain of streptococcalprotein G and comparison with NMR. Biochemistry 19, 4721-4729.

Reference 4; Sauer-Eriksson AE, Kleywegt GJ, Uhlen M, Jones TA. (1995) Crystal structure of the C2 fragment of streptococcal protein G in complex with the Fc domain of human IgG. Structure 3, 265-278.

Reference 5; Derrick JP, Wigley DB. (1994) The third IgG-binding domain from streptococcal protein G. An analysis by X-ray crystallography of the structure alone and in a complex with Fab. J Mol Biol. 243, 906-918.

Reference 6; Alexander P, Fahnestock S, Lee T, Orban J, Bryan P. (1992) Thermodynamic analysis of the folding of the streptococcal protein G IgG-binding domains B1 and B2: why small proteins tend to have high denaturation temperatures. Biochemistry 14, 3597-3603.

Reference 7; D'souza VM, Holz RC. (1999) The methionyl aminopeptidase from Escherichia coli can function as an iron(II) enzyme. Biochemistry 38, 11079-11085.

Reference 8; Horton R. M., Hunt H. D., Ho S. N., Pullen J. M. and Pease L.R. (1989). Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77, 61-68.

Reference 9; Okada Masato, Miyazaki Ka (2004) Protein Experiment Notes (Part 1), Yodosha.

Reference 10; Edited by Shigeo Ohno and Yoshifumi Nishimura (1997) Protein Experiment Procedure 1-Functional Analysis, edited by Shujunsha.

Reference 11; Edited by Shigeo Ohno and Yoshifumi Nishimura (1997) Protein Experiment Procedure 2-Structure Analysis, edited by Shujunsha.

In this specification, various amino acid residues are described with the following abbreviations. Ala; L-alanine residue, Arg; L-arginine residue, Asp; L-aspartic acid residue, Asn; L-asparagine residue, Cys; L-cysteine residue L-glutamine residue, Glu; L-glutamic acid residue, Gly; L-glycine residue, His; L-histidine residue, Ile; L-isoleucine residue , Leu; L-leucine residues, Lys; L-lysine residues, Met; L-methionine residues, Phe; L-phenylalanine residues, Pro; L-proline L-serine residue, Thr; L-threonine residue, Trp; L-tryptophan residue, Tyr; L-tyrosine residue, Val; L-valine residue . In addition, in this specification, the amino acid sequence of a peptide is described according to a conventional method such that the amino terminal (hereinafter referred to as N-terminal) is on the left and the carboxy-terminal (hereinafter referred to as C-terminal) is on the right.

More specifically, Patent Document 8 specifically discloses the following.

Example 1 describes the wild-type amino acid sequence based on the protein G・B1 domain shown in [SEQ ID NO:1] and the wild-type amino acid sequence of the protein G・B2 domain shown in [SEQ ID NO:2] and the wild-type amino acid sequence of the protein G·B3 domain shown in [SEQ ID NO: 3], selected for the design of introducing mutations into the B1, B2, or B3 domain of protein G and used in the present invention In the amino acid sequence of the mutant protein contained in the protein (hereinafter referred to as "modified protein G"), the mutation-introduced site specifically specifies the amino acid residue to be substituted.

In Example 2, it is described that multiple modified proteins G shown in [SEQ ID NO:4] to [SEQ ID NO:19] are designed by using the information of the selected mutation target site and the above-mentioned specific substituted amino acid residues Further, [SEQ ID NO: 13] ~ [SEQ ID NO: 20] were finally selected as the specific amino acid sequence, and the modified protein G showing the sequence was actually synthesized, and its molecular characteristics were evaluated.

Example 3 describes the base sequence ([SEQ ID NO: 13] to [SEQ ID NO: 20]) of the nucleic acid encoding the amino acid sequence of modified protein G and the c-terminal of the α-subunit of oxaloacetate decarboxylase Base sequence of structural domain (OXADac) [SEQ ID NO: 31] The base sequence of the mutant protein.

It is described in Example 4 that a plasmid vector containing a gene encoding modified protein G was synthesized by using the PG gene composed of the base sequence of [SEQ ID NO:21] to [SEQ ID NO:29], and then using Escherichia coli to A fusion protein of oxaloacetate decarboxylase α-subunit c-terminal domain (OXADac) [SEQ ID NO:31] and mutant protein was prepared.

Example 5 describes the use of various primers ([SEQ ID NO: 32] to [SEQ ID NO: 35]) to synthesize a plasmid vector containing a gene encoding modified protein G, and then use Escherichia coli to prepare Met to add modified Protein G.

Described in embodiment 6 confirms the purity of modified protein G with polyacrylamide gel electrophoresis; Described in embodiment 7 by measuring the protein prepared by the molecular weight identification of modified protein G with MALDI-TOF type mass spectrometer; Implement Example 8 describes the evaluation of antibody dissociation of modified protein G in a weakly acidic region by using a column of immobilized OXADac-PG fusion protein to perform pH gradient affinity chromatography and study the pH of monoclonal antibody elution; Example 9 describes the evaluation of the antibody dissociation of modified protein G in the weakly acidic region by using a column of immobilized OXADac-PG fusion protein for stepwise pH affinity chromatography and studying the elution of monoclonal antibodies at several pHs; It is described in Example 10 that the binding and dissociation properties of mutant proteins (protein G mutants) are evaluated by the surface plasmon resonance (SPR) method; In the weakly acidic region protonated above %, the antibody binding of the mutant protein was evaluated by the surface plasmon resonance (SPR) method; the thermal stability of the mutant protein was evaluated in Example 12; the prepared The single crystal of the mutant protein was analyzed by X-ray diffraction to determine the three-dimensional structure.

Further, Example 14 describes the use of a trimeric wild-type PG (CGB01H-3D, SEQ ID NO: 36) that incorporates a cysteine residue and a His tag that encodes the addition of a cysteine residue at the carboxy-terminal side or as the protein of the present invention. Two kinds of artificially synthesized plasmids of the tandem trimer (CGB19H-3D, SEQ ID NO: 37) gene of the mutant PG, to prepare the tandem multimer of the cell membrane domain mutant of protein G and make it for use The affinity chromatography column of the protein and the purification of human IgG1 antibody or IgG3 antibody etc. by using the column.

Example 15 describes the comparison of tandem multimers and homomonomers of protein G extracellular domain mutants with respect to antibody binding and dissociation properties of IgG1-type humanized monoclonal antibodies.

Example 16 describes the use of a monomeric mutant PG (CGB19H-1D, FIG. 4 , SEQ ID NO: 38) incorporating a mutant PG encoding a His tag added at the carboxy-terminal cysteine residue, mutation The gene of the tandem tetramer PG (CGB19H-4D, Fig. 4, SEQ ID NO:39) of type PG and the tandem pentamer PG (CGB19H-5D, Fig. 4, SEQ ID NO: 40) of mutant PG Three kinds of artificially synthesized expression plasmids were used to prepare monomers, tandem tetramers and pentamers of protein G extracellular domain mutants.

In Example 17, it is described that the monomers and tandem polymers of protein G extracellular domain mutants were immobilized on a solid phase via the carboxyl-terminal cysteine residue, and the mutant proteins were compared and evaluated by the SPR method. Antibody binding to humanized monoclonal antibodies of IgG1 type.

Hereinafter, the present invention will be specifically described using examples. However, the technical scope of the present invention is not limited to these Examples.

Example 1

Preparation of tandem dimers of extracellular domain mutants of protein G

In this example, the tandem dimers of protein G extracellular domain mutants with different linker lengths of the present invention were prepared.

A 60-residue 10-repeat unit of 6 residues consisting of glycine and serine that encodes the addition of a cysteine residue and a His tag to the carboxyl terminus and contains a restriction enzyme recognition site between the domains was synthesized Plasmid for expression of linker dimer mutant PG (PG2LL10, FIG. 1 , SEQ ID NO:41) gene. Next, based on the method of Evers et al. (Non-Patent Document 9), dimeric mutant PGs (PG2LL7, PG2LL6, PG2LL7, PG2LL6, , PG2LL5, PG2LL4, PG2LL1, Figure 1, the expression plasmids of SEQ ID NO: 42-46). Expression Escherichia coli BL21(DE3) strain (Novagen) was transformed with dimer mutant PG plasmids of various linker lengths. Passage the pre-cultured transformant in 2YT medium at 2ml/200ml, shake culture until OD ₆₀₀ =0.8～1.0. In order to express the target protein, 0.5 mM IPTG was added, and further cultured with shaking at 37° C. for 2 hours. The recovered bacterial cells were suspended in 10 mL of PBS, ultrasonically disrupted, and then sterilized by filtration, which was used as a complete protein solution. Recombinant PG was adsorbed on a Ni Sepharose (GE Healthcare) 2 ml column, washed with 20 mM imidazole, and eluted with 500 mM imidazole to obtain a purified protein.

Example 2

Evaluation of Antibody Binding of Protein G Extracellular Domain Mutant Tandem Dimer

In this example, the tandem dimer of the protein G extracellular domain mutant (SEQ ID NO: 19) with different linker lengths of the present invention was immobilized on a solid via the cysteine residue at the carboxyl terminal Phase, using the SPR method to compare and evaluate the antibody binding of each mutant protein.

First, the protein G extracellular domain mutant tandem dimers (PG2LL1, PG2LL4, PG2LL5, PG2LL6, PG2LL7, PG2LL10) prepared in Example 1 with different linker lengths were each prepared using EMCH (N-[ε- Maleimide caproic acid]hydrazide, trifluoroacetic acid) (Thermo scientific) was immobilized to the assay cell of the sensor chip CM-5 (GE Healthcare) by maleimide coupling. The amount of immobilization was done in such a way that the same mass of protein was immobilized. Next, humanized monoclonal antibody of IgG1 type was dissolved in running buffer (10 mM HEPES pH7.4, 150 mM NaCl, 0.005% v/v surfactant P20), and adjusted to 10 μM and 1 μM sample solutions. Using Biacore T100 (GE Healthcare), SPR measurement was carried out at a reaction temperature of 25° C. ( FIG. 2-1 , FIG. 2-2 ).

Let the sample antibody flow through the chip on which the extracellular domain mutant tandem dimer of protein G with different linker lengths of the present invention is immobilized with the same mass. After 500 seconds, measure the amount of antibody bound to the chip. As a result, observe The antibody binding rate increased as the linker length increased, and the maximum antibody binding rate was seen on the chip immobilized with a dimer (PG2LL7) containing a 42-residue linker ( FIG. 3 ). In addition, it was observed that the concentration of the antibody in the sample decreased and the difference in the binding rate of the antibody among the linker lengths became larger ( FIG. 3 ). From the above, it can be seen that the tandem dimer of the present invention has a linker length suitable for efficiently binding the sample antibody.

Example 3

Preparation of extracellular domain mutants of protein G and fabrication of columns using the protein

(1) Expression and purification of recombinant PG

Among the mutants constructed in Example 1, PG2LL7 and rPG-A4 described in Example 16 of Patent Document 8, which is a tandem tetramer PG with a cysteine residue added to the carboxy terminus and a His tag, were used. (CGB19H-4D, SEQ ID NO: 39) The transformed Escherichia coli was subcultured in LB medium and cultured with shaking until O.D.600 = 0.8-1.0. In order to express the target protein, 0.5 mM IPTG was added, and further cultured with shaking at 37° C. for 3 hours. The recovered bacterial cells were suspended in PBS, subjected to ultrasonic disruption and centrifuged, and the obtained supernatant was used as a complete protein solution. Recombinant PG was adsorbed on a HisTrap FF (GE Healthcare Bioscience) 1 ml column, washed with 20 mM imidazole, and eluted with 500 mM imidazole to obtain a purified protein.

(2) Immobilization and column preparation of recombinant PG

Sepharose4FastFlow (GE Healthcare) was separated by filtration with a glass filter and washed with ultrapure water to obtain 10 ml of the carrier. The carrier was transferred to a flask, 3 ml of a 2M aqueous sodium hydroxide solution and 4 g of butanediol diglycidyl ether were added, and the mixture was shaken at 25° C. for 4 hours to perform a reaction. It was separated by filtration with a glass filter and washed with ultrapure water to obtain an activated carrier. 1 mL of the activated carrier was collected into a glass filter and washed with a coupling buffer (0.1 M sodium phosphate, 1.0 M sodium sulfate, 1 mM EDTA, pH 8.0). Move the activated carrier to a flask, add 1.5 ml of a recombinant PG (PG2LL7) solution containing 5.4 mg/ml recombinant PG, 2 mL of a coupling buffer, shake at 150 rpm at 37°C for 25 hours to pass through the cysteine of the recombinant PG Acid residues are immobilized. Isolate by filtration with a glass filter and wash with coupling buffer. Next, the carrier was transferred to a flask, 3 ml of a solution of 1 M thioglycerol, 0.1 M sodium phosphate, 1 mM EDTA, pH 8.0 was added, and the unreacted active groups were shielded by shaking at 150 rpm at 37° C. for 4 hours. Filter and separate with a glass filter, wash 3 pieces alternately with cleaning solution 1 (0.1M Tris hydrochloric acid, 0.5M sodium chloride, pH8.0) and cleaning solution 2 (0.1M acetic acid, 0.5M sodium chloride, pH4.0) 15ml cycle. Wash 1 ml of the immobilized carrier with ultrapure water and fill it into a Tricon 5/50 column. In addition, rPG-A4 was also prepared by the same operation as that of PG2LL7.

Example 4

Antibody Adsorption Capacity of Recombinant PG Immobilized Column

Install the recombinant PG immobilized column made in Example 3 on the liquid chromatography device AKTAexplore (GE Healthcare Bioscience), make the adsorption buffer (20mM phosphate buffer, 150mM sodium chloride, pH7.2) at 1mL/min or 0.4 After equilibrating by flowing under the condition of mL/min, human IgG (Oriental yeast) prepared at 1 mg/mL was injected. The injection was continued until the absorbance at 280 nm of the eluate reached 15% of the absorbance of the injected sample, and then, after washing with the adsorption buffer, the adsorption buffer was replaced with 20 mM citric acid (pH 2.4).

Dynamic binding capacity (DBC) was calculated from the amount of sample injected until the absorbance of non-adsorbed components at 280 nm excluding the eluate reached 10% of the absorbance of the injected sample. The DBC of each immobilized column is shown in Table 1. It was found that PG2LL7 and rPG-A4 exhibit substantially the same DBC, showing high binding capacity by extending the linker part, appropriately controlling the distance between the antibody-binding domains at both ends instead of inserting the antibody-binding domains.

[Table 1]

Example 5

pH gradient affinity chromatography

The recombinant PG immobilized column was installed in the liquid chromatography device AKTAexplore (GE Healthcare Bioscience), and the adsorption buffer (20mM phosphate buffer, 150mM sodium chloride, pH7.2) was flowed under the condition of 1 ml/min to balance the After incubation, human IgG (Oriental yeast) prepared at 1 mg/mL was injected. After washing with adsorption buffer, it was replaced with 20 mM citric acid buffer (pH 6.0), and continuously replaced with 20 mM citric acid (pH 2.4) at a flow rate of 1.0 mL/min for 80 min.

The human IgG elution peak was around pH 3.9 in the PG2LL7 immobilized column, and around pH 4.4 in the rPG-A4 immobilized column (Fig. 4). It can be seen that the PG2LL7 immobilized column has the same DBC as rPG-A4, and can be eluted under milder acidic conditions.

Industrial Applicability

Currently, the extracellular domain of wild-type protein G is commercially available as an affinity chromatography support for antibody purification or a test reagent for antibody detection, and is widely used in various fields of life sciences. In addition, with the development of antibody-related industries such as antibody medicine in recent years, the demand for these products has expanded dramatically. Therefore, instead of protein G and protein A, use the protein of the present invention that has at least the same binding activity in the neutral region as conventional proteins and can dissociate and elute immunoglobulins under milder conditions in the weakly acidic region. The wild-type protein and the conventional mutant protein (modified protein) containing its domain mutants can reduce the degradation of antibodies accompanying acid elution, and are widely used in the field of processing antibodies derived from various animal species , greatly contribute to its technological development.

Claims (27)

1. A protein formed by bonding a plurality of structural domains having affinity for a protein having an Fc portion of immunoglobulin G (IgG) with a linker, wherein the linker has a maximum elongation length of 80 Å to 240Å. 2. The protein of claim 1, wherein the linker is a polypeptide linker. 3. The protein according to claim 2, wherein the linker consists of a polypeptide of 22-66 amino acid residues. 4. The protein according to claim 3, wherein the amino acid sequence of the linker consists of 4-10 repeating units of GlyGlySerGlyGlySer. 5. The protein according to claim 4, wherein the amino acid sequence of the linker consists of 6-10 repeating units of GlyGlySerGlyGlySer. 6. The protein according to any one of claims 1 to 5, wherein the domain is derived from protein G or protein A. 7. The protein according to claim 6, wherein the structural domain is a mutant of any one of the wild-type protein G·B1, B2, or B3 domains. 8. The protein according to any one of claims 1 to 7, wherein the domains are identical to each other. 9. The protein according to any one of claims 1 to 8, wherein the two domains are a linked dimer. 10. The protein according to any one of claims 7 to 9, wherein the domain mutant is a mutant protein of: A mutant protein of the wild-type protein G·B1 domain protein, which consists of the amino acid sequence shown in (a) below, or one or more amino acid residues in the amino acid sequence shown in (a) are deleted, Substituted, inserted or added amino acid sequence composition, has binding activity to the Fc region of immunoglobulin G, and at least has binding activity to the Fab region of immunoglobulin G and / or reduced binding activity to the Fc region in the weakly acidic region: (In the above amino acid sequences, X35 represents Asn or Lys, X36 represents Asp or Glu, X37 represents Asn, His, or Leu, X47 represents Asp or Pro, X48 represents Ala, Lys or Glu, X22 represents Asp or His, respectively, in the above amino acid sequences, X25 represents Thr or His, X32 represents Gln or His, X40 represents Asp or His, X42 represents Glu or His, X11 represents Thr or Arg, X17 represents Thr or Ile, however, X35 is Asn or Lys, X36 is Asp simultaneously or Glu, X37 is Asn or Leu, X47 is Asp or Pro, X48 is Ala, Lys or Glu, X22 is Asp, X25 is Thr, X32 is Gln, X40 is Asp, X42 is Glu, X11 is Thr, and X17 is In the case of Thr). 11. The protein according to any one of claims 7 to 9, wherein the domain mutant is a mutant protein of: A mutant protein of the wild-type protein G.B2 domain protein, which consists of the amino acid sequence shown in (b) below, or one or more amino acid residues in the amino acid sequence shown in (b) are deleted, Substituted, inserted or added amino acid sequence composition, has binding activity to the Fc region of immunoglobulin G, and at least has binding activity to the Fab region of immunoglobulin G and / or reduced binding activity to the Fc region in the weakly acidic region: (In the above amino acid sequences, X35 represents Asn or Lys, X36 represents Asp or Glu, X37 represents Asn, His, or Leu, X47 represents Asp or Pro, X48 represents Ala, Lys or Glu, X22 represents Asp or His, respectively, in the above amino acid sequences, X25 represents Thr or His, X32 represents Gln or His, X40 represents Asp or His, X42 represents Glu or His, X11 represents Thr or Arg, X17 represents Thr or Ile, however, X35 is Asn or Lys, X36 is Asp simultaneously or Glu, X37 is Asn or His, X47 is Asp or Pro, X48 is Ala, Lys or Glu, X22 is Asp, X25 is Thr, X32 is Gln, X40 is Asp, X42 is Glu, X11 is Thr, and X17 is In the case of Thr). 12. The protein according to any one of claims 7 to 9, wherein the domain mutant is a mutant protein of: A mutant protein of the wild-type protein G.B3 domain protein, which consists of the amino acid sequence shown in (c) below, or one or more amino acid residues in the amino acid sequence shown in (c) are deleted, Substituted, inserted or added amino acid sequence composition, has binding activity to the Fc region of immunoglobulin G, and at least has binding activity to the Fab region of immunoglobulin G and / or reduced binding activity to the Fc region in the weakly acidic region: (In the above amino acid sequences, X35 represents Asn or Lys, X36 represents Asp or Glu, X37 represents Asn, His, or Leu, X47 represents Asp or Pro, X48 represents Ala, Lys or Glu, X22 represents Asp or His, respectively, in the above amino acid sequences, X25 represents Thr or His, X32 represents Gln or His, X40 represents Asp or His, X11 represents Thr or Arg, X17 represents Thr or Ile, however, X35 is Asn or Lys, X36 is Asp or Glu, X37 is Asn or His, X47 is Asp or Pro, X48 is Ala, Lys or Glu, X22 is Asp, X25 is Thr, X32 is Gln, X40 is Asp, X11 is Thr, and X17 is Thr). 13. The protein according to any one of claims 7 to 9, wherein the domain mutant is a mutant protein of: A mutant protein of the wild-type protein G·B1 domain protein, which consists of the amino acid sequence shown in (d) below, or one or more amino acid residues in the amino acid sequence shown in (d) are deleted, Substituted, inserted, or added amino acid sequences have binding activity to the Fc region of immunoglobulin G, and, compared with wild-type protein G·B1 domain protein, binding activity to the Fab region of immunoglobulin G and / or reduced binding activity to the Fc region in the weakly acidic region: (in the above amino acid sequences, respectively, X22 represents Asp or His, X25 represents Thr or His, X32 represents Gln or His, X40 represents Asp or His, X42 represents Glu or His, X11 represents Thr or Arg, X17 represents Thr or Ile , however, exclude the case where X22 is Asp, X25 is Thr, X32 is Gln, X40 is Asp, X42 is Glu, X11 is Thr, and X17 is Thr simultaneously). 14. The protein according to any one of claims 7 to 9, wherein the domain mutant is a mutant protein of: A mutant protein of the wild-type protein G·B2 domain protein, which consists of the amino acid sequence shown in (e) below, or one or more amino acid residues in the amino acid sequence shown in (e) are deleted, Substituted, inserted or added amino acid sequence composition, has binding activity to the Fc region of immunoglobulin G, and has binding activity to the Fab region of immunoglobulin G and/or compared with wild-type protein G·B2 domain protein Or the binding activity of the Fc region in the weakly acidic region is reduced: (in the above amino acid sequences, respectively, X22 represents Asp or His, X25 represents Thr or His, X32 represents Gln or His, X40 represents Asp or His, X42 represents Glu or His, X11 represents Thr or Arg, X17 represents Thr or Ile , however, exclude the case where X22 is Asp, X25 is Thr, X32 is Gln, X40 is Asp, X42 is Glu, X11 is Thr, and X17 is Thr simultaneously). 15. The protein according to any one of claims 7 to 9, wherein the domain mutant is a mutant protein of: A mutant protein of the wild-type protein G.B3 domain protein, which consists of the amino acid sequence shown in (f) below, or one or more amino acid residues in the amino acid sequence shown in (f) are deleted, Substituted, inserted or added amino acid sequence composition, has binding activity to the Fc region of immunoglobulin G, and has binding activity to the Fab region of immunoglobulin G and/or compared with the wild-type protein G·B3 domain protein Or the binding activity of the Fc region in the weakly acidic region is reduced: (In the above amino acid sequences, X22 represents Asp or His, X25 represents Thr or His, X32 represents Gln or His, X40 represents Asp or His, X11 represents Thr or Arg, X17 represents Thr or Ile, respectively, but excludes simultaneously X22 is Asp, X25 is Thr, X32 is Gln, X40 is Asp, X11 is Thr, and X17 is Thr). 16. The protein according to any one of claims 7 to 9, wherein the domain mutant is a mutant protein of: A mutant protein of the wild-type protein G·B1 domain protein, which consists of the amino acid sequence shown in (g) below, or one or more amino acid residues in the amino acid sequence shown in (g) are deleted, Substituted, inserted or added amino acid sequences have binding activity to the Fc region of immunoglobulin G, and compared with the wild-type protein G B1 domain protein, the binding activity to the Fc region in the weakly acidic region is reduced: (In the above amino acid sequences, respectively, X22 represents Asp or His, X25 represents Thr or His, X32 represents Gln or His, X40 represents Asp or His, X42 represents Glu or His, however, X22 is Asp and X25 is Thr, X32 is Gln, X40 is Asp, and X42 is Glu). 17. The protein according to any one of claims 7 to 9, wherein the domain mutant is a mutant protein of: A mutant protein of the wild-type protein G.B2 domain protein, which consists of the amino acid sequence shown in (h) below, or one or more amino acid residues in the amino acid sequence shown in (h) are deleted, Substituted, inserted or added amino acid sequences have binding activity to the Fc region of immunoglobulin G, and compared with the wild-type protein G B2 domain protein, the binding activity to the Fc region in the weakly acidic region is reduced: (In the above amino acid sequences, respectively, X22 represents Asp or His, X25 represents Thr or His, X32 represents Gln or His, X40 represents Asp or His, X42 represents Glu or His, however, X22 is Asp and X25 is Thr, X32 is Gln, X40 is Asp, and X42 is Glu). 18. The protein according to any one of claims 7 to 9, wherein the domain mutant is a mutant protein of: A mutant protein of the wild-type protein G.B3 domain protein, which consists of the amino acid sequence shown in (i) below, or one or more amino acid residues in the amino acid sequence shown in (i) are deleted, Substituted, inserted or added amino acid sequences have binding activity to the Fc region of immunoglobulin G, and compared with the wild-type protein G B3 domain protein, the binding activity to the Fc region in the weakly acidic region is reduced: (In the above amino acid sequences, respectively, X22 represents Asp or His, X25 represents Thr or His, X32 represents Gln or His, and X40 represents Asp or His, however, simultaneously X22 is Asp, X25 is Thr, X32 is Gln, and X40 is the case for Asp). 19. The protein according to any one of claims 7 to 9, wherein the domain mutant is a mutant protein having the following amino acid sequence (SEQ ID NO: 19): . 20. A protein which is a fusion protein consisting of an amino acid sequence linking the amino acid sequence of the protein according to any one of claims 1 to 19 and the amino acid sequence of another protein. 21. The protein according to claim 20, which has the amino acid sequence shown in any one of SEQ ID NO: 41-45. 22. A nucleic acid encoding the protein of any one of claims 1-21. 23. A recombinant vector comprising the nucleic acid of claim 22. 24. A transformant into which the recombinant vector according to claim 23 has been introduced. 25. A capture agent for immunoglobulin G or a protein having an Fc region or a Fab region of immunoglobulin G, wherein the protein according to any one of claims 1 to 21 is immobilized on a water-insoluble solid phase support body. 26. Affinity chromatography for purification of immunoglobulin G or a protein having an Fc region or Fab region of immunoglobulin G, comprising the capture agent according to claim 25. A method for purifying immunoglobulin G or a protein having an Fc region or Fab region of immunoglobulin G, using the affinity chromatography for purification according to claim 26.